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Intracellular Loop 2 Peptides of the Human 5HT1a Receptor are Differential Activators of Gi

Intracellular Loop 2 Peptides of the Human 5HT1a Receptor are Differential Activators of Gi Hindawi Publishing Corporation International Journal of Peptides Volume 2012, Article ID 490734, 8 pages doi:10.1155/2012/490734 Research Article Intracellular Loop 2 Peptides of the Human 5HT1a Receptor are Differential Activators of Gi Brian Hall, Carley Squires, and Keith K. Parker Department of Biomedical and Pharmaceutical Sciences (MPH I02), Center for Structural and Functional Neuroscience, Skaggs School of Pharmacy, The University of Montana, 32 Campus Drive No. 1552, Missoula, MT 59812-1552, USA Correspondence should be addressed to Keith K. Parker, keith.parker@umontana.edu Received 27 December 2011; Revised 10 February 2012; Accepted 24 February 2012 Academic Editor: Piero Andrea Temussi Copyright © 2012 Brian Hall et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Peptide mimics of intracellular loop 2 (ic2) of the human 5HT1a receptor have been studied with respect to their ability to inhibit agonist binding via interference with receptor-G-protein coupling. These peptides give shallow concentration-effect relationships. Additionally, these peptides have been studied with respect to their ability to trigger the signal transduction system of this Gi-coupled receptor. Two signaling parameters have been quantified: concentration of intracellular cAMP and changes in incorporation into the G protein of a stable analog of GTP. In both cases, peptide mimics near midloop of ic2 actually show agonist activity with efficacy falling off toward both loop termini near TM 3 and TM 4. Previous results have suggested that the loop region near the TM3/ic2 interface is primarily responsible for receptor-G-protein coupling, while the current result emphasizes the mid- ic2 loop region’s ability to activate the G protein following initial coupling. A limited number of peptides from the receptor’s TM5/ic3 loop vicinity were also studied regarding agonist inhibition and G-protein activation. These peptides provide additional evidence that the human 5HT1a receptor, TM5/ic3 loop region, is involved in both coupling and activation actions. Overall, these results provide further information about potential pharmacological intervention and drug development with respect to the human 5HT1a receptor/G-protein system. Finally, the structural evidence generated here provides testable models pending crystallization and X-ray analysis of the receptor. 1. Introduction greater fundamental knowledge but to therapeutic develop- ment. Regulation of serotonergic (5-hydroxytryptamine; 5HT) Various receptors (R), including the 5HT3R’s that are function in animals impacts numerous physiological and ligand-controlled ion channels, are crucial to these regula- pathological processes [1]. 5HTisbroadly representedinbi- tory processes [4]. All other known 5HTR’s are structurally ological systems as a regulator and modulator via nervous, different than these ion channels, being serpentine mem- hormonal, and autacoidal means [2–5]. For example, sero- brane R’s [8], coupled (C) to the cells interior by G (GTP tonin [6] has been implicated in the pathophysiology of binding) proteins (P), which in turn regulate key effectors migraine. This association with migraine is shared with many such as adenylyl cyclase (AC, [9]). These GPCR’s share the other factors including adipokines such as leptin; hypothala- structural characteristic of 7-transmembrane (7TM) helical mic hormones, Orexin A and B (also known appetite regula- segments [10–13]. For many years, the only crystal structure tors as is 5HT); numerous neurotransmitters [7]; autacoids; was of rhodopsin, the prototype GPCR, in its interaction hormones, and ions like calcium, and magnesium. The with the G-protein transducin [14]. Recently a breakthrough range of biological molecules that interact with serotonergic has occurred, with crystallization of the beta-adrenergic processes suggests that various signaling pathways may be receptor (BAR) and publication of X-ray structures [15– shared, and that the potential for dynamic, collaborative 18]. This long-awaited event has set the stage for other regulation exists. Better understanding of the molecular basis GPCR. Progressive developments have been demonstrated by underlying these signaling processes is not only critical to crystal structures for the adenosine A2R [19], the CXDR4 2 International Journal of Peptides chemokine R [20], and the dopamine D3R [21]. Crystal Reactions were stopped by addition of 4 mL of ice-cold structures for other GPCR, including that those 5HTR’s that 50 mM Tris buffer, pH 7.4, and vacuum filtration on glass are GPCR’s should soon follow [22]. fiber filters (Whatman GF/B). Filters were rinsed twice in Of the GPCR recognized as 5HTR, the 5HT1aR (a relative 5 mL of ice-cold Tris buffer, dried, and counted in 5 mL of of BAR) is one of the most highly studied [23–25], and Ecoscint (National Diagnostics) liquid scintillation fluid in a it has been associated with physiological and pathological Beckman LS 6500. Homogenates were assayed for protein to processes as diverse as thermoregulation, cognitive flexibility, maintain a nominal value of 50 μg protein per filter [57]. All and control of mood [26–33]. Depression, underlying anxi- tubes were run in triplicate. ety disorders, and related psychopathologies are a particular theme [34–39]. Multiple strategies have been used to dissect 2.4. cAMP Assay. CHO cells were cultured to confluency in the complex pathways underscoring these physiological 12- or 24-well plates. Medium was aspirated, and the cells and pathological processes [40–47]. One approach centers were rinsed twice in warm, serum-free F-12 medium. Cells around analysis of allosteric sites of action on receptors. were incubated for 20 min. at 37 Cin0.5mlsofserum-free Peptide mimics of intracellular loop regions of 5HT1aR have F-12 medium containing 100 uM isobutylmethylxanthine been used as probes of the receptor-G-protein interface in (IBMX) and the following substances (final concs.) alone or this context [48–54]. The current communication continues in combination (see Figures 3 and 5): 30 μM forskolin (FSK; our analysis with these peptide probes particularly emphasiz- for all treatments); 1 μM 5-HT; peptide concentrations as ing intracellular loop 2 (ic2) with some, limited comparative noted in figure legends. Reactions were stopped by aspiration data from intracellular loop 3 (ic3). The results with ic2 of medium and addition of 0.5 mL of 100 mM HCl. After and ic3 are suggestive of potential sites for regulation and 10 min., well contents were removed and centrifuged at therapeutic drug development. 4000 rpm. Supernatants were diluted in 100 mM HCl, and cAMP was quantified [53] directly in a microplate format by enzyme immunoassay (EIA) with a kit from Assay Designs 2. Materials and Methods (Ann Arbor). Triplicate-independent samples were assayed. 2.1. Cell Culture. Chinese Hamster ovary (CHO) cells ex- 2.5. [35S]GTPγS Assay. H5-HT1aR membranes from trans- pressing the H5-HT1aR [55, 56]wereculturedinHam’s F-12 fected CHO cells were incubated with 5-HT (0.1 μM) and/or medium fortified with 10% fetal calf serum and 200 ug/mL peptide concentrations as noted in figure legends (see geneticin. Cultures were maintained at 37 C in a humidified Figures 2 and 4) and the following incubation mixture: atmosphere of 5% CO . Cells were subcultured or assayed 20 mM HEPES buffer, pH 7.4, 5 mM MgCl ,1mM EDTA, upon confluency (5–8 days). Cloned H5-HT1aR was kindly 2 1 mM DTT, 100 mM NaCl, 100 uM GDP, 10 μM pargyline, provided by Dr. John Raymond (Medical U. of South 0.2 mM ascorbate, and 0.1 nM [35S]GTPγS[53, 58]. Mix- Carolina; [41]). The cell line has been tested for mycoplasma tures were incubated for 30 min. at 30 C, and were terminat- with a PCR kit (ATCC) and is free of contamination. ed by dilution in cold buffer. The mixture was filtered on GF/C filters, rinsed twice in buffer, dried and counted by 2.2. Receptor Preparation. Cells were trypsinized and cen- liquid scin-tillation. All values reported in are for specific trifuged at low speed in ice-cold medium [53]. The pellet binding (total nonspecific) of triplicates. Nonspecific bind- was resuspended in ice-cold Earle’s Balanced Salt Solution ing was determined in the presence of cold γ-S-GTP (10 uM). followed by centrifugation. Cells were resuspended in 10 mL Negative controlisthe abovemixture minustestdrugor of ice-cold binding buffer (50mM Tris, 4mM CaCl ,10 μM 5HT. Positive control contains 5HT. pargyline, and pH 7.4), homogenized with Teflon-glass, and centrifuged for 450,000 g-min. at 4 C. For a crude membrane 2.6. Data Analysis. All statistics (means, standard errors of preparation, the pellet was resuspended in 30 mL of ice-cold the mean (SEM), t tests and ANOVA, Pearson correlation binding buffer, homogenized on Teflon-glass and then by coefficients (r), and graphical procedures (including drug- Polytron (setting 4) for 5 seconds, and stored on ice and receptor-binding analysis) were conducted with PSI-Plot assayed within the next 1.5 hours [54]. (Version 8) software (Poly Software International), Prism (version 4.0c), or using a Hewlett-Packard Graphing Calcu- lator, HP48. The apriori was α = 0.05 for all experiments. 2.3. Assay of Receptor Activity. Binding of the agonist [3H]8- Experiments were conducted with a minimum of n = 3, in OH-DPAT ([3H]8-hydroxy-2-(di-n-propylamino)tetralin) triplicate. Most experiments were n = 3–5. In some cases to H5-HT1aR followed well-characterized protocols (indicated in figure legends), different n’s and multiplicates [49, 50, 53]. Radioligands were purchased from New England were used. Nuclear (NEN), Boston, MA, and 1 mL reaction mixtures, in triplicate, were incubated for 30 min. in a 30 C shaker. The 1 mL mixture was 700 μLofreceptorpreparation; 2.7. Peptide Preparation. These highly purified (greater than 100 μL of binding buffer (for total binding) or 10 μM 5-HT 95%) peptides were purchased from New England Peptide (for nonspecific binding), 100 μL of the tritiated agent LLC. The peptides are segments of ic2 and ic3 of the cloned (concentration of 0.5 nM [3H] 8-OH-DPAT), and 100 μLof H5HT1aR. Peptides stored at −20 C were initially dissolved peptide or binding buffer in the case of controls. in deionized water. Subsequent dilutions were in binding International Journal of Peptides 3 Table 1: ic2 and ic3 peptide mimics. The primary amino acid seq- uences for the H5HT1aR ic2 loop peptide mimics P11 and P’s 21– 120 27, and for ic3 (P1, P12, and P13). The receptor’s amino terminal is to the left. Sequences for H5HT1aR from Kobilka et al., 1987 [56]. P11 is from a previous study by Thiagaraj et al., 2007 [53], and P1 from Hayataka et al., 1988 [49] (both included for comparative purposes). 0 5 10 15 20 25 30 35 P11 IALDRYWAITD P21 LDRYWAITDP P21 concentration μM P22 RYWAITDPID Figure 1: P21 noncentration-dependent displacement of bound 8- P23 WAITDPIDYV OH-DPAT. This curve represents the change in specific binding of P24 ITDPIDYVNK [ H]-8-OH-DPAT, a 5HT1aR agonist, to the receptor in the pres- ence of various concentrations of the ic2 peptide mimic P21. Nom- P25 DPIDYVNKRT inal binding of agonist at control levels was 400 fmoles/mg protein. P26 IDYVNKRTPR P27 YVNKRTPRPR P1 IFRAARFRIRKTVKK Peptide effect on γ-[ S]-GTP P12 KTVKKVEKTG incorporation P13 VKKVEKTGAD buffer. The peptides examined in this study are listed in Table 1. Control 5HT P21 P22 P23 P24 P25 P26 P27 3. Results Figure 2: ic2 peptide effect on γ-[ S]-GTP incorporated into Gi, 3.1. Intracellular Loop 2 (ic2). ThesizeofH5HT1a’sic2 a measure of G-protein activation. Control is the basal amount (about 20 amino acids) makes it a tempting target for analyz- 35 of γ-[ S]-GTP incorporated into Gi in CHO cells expressing ing the loop’s coupling to and activation of Gi [23, 24]. Our the human 5HT1aR, set as 100%. The Y-axisisthe percentof previous work with ic2 emphasized the N-terminal region of specifically bound (total minus nonspecific) γ-[ S]-GTP. All other the loop with a peptide we call P11 (Table 1). Results with treatments are percents of the control value. All peptides are 30 uM −7 concentration and 5HT 10 M concentration. Error is expressed as this peptide suggest that the loop residues near TM 3 are SEM. vital for coupling of the loop to Gi but are not involved in G- proteinactivation[53]. Results from the Varrault group [48] looked at the entire loop without distinguishing subregions; their conclusions were that the entire loop is responsible perturb G protein following coupling. The overall results for for activation (they did not differentiate between coupling these eight peptides are in Table 2. and activation). The following question arises: can coupling As shown in Figure 1 with results from peptide P21 as and activation characteristics be identified for the loop an example, these peptides give shallow concentration-effect on a subregional basis? Our preliminary work at the N- curves for the measure of coupling and agonist inhibition. terminal aspect of H5HT1a’a ic3 suggested to us that the Similar experiments with all peptides form the basis for techniques we use could be productive in addressing such a the summarized coupling results found in Table 2.Note question [49, 50, 53]. Thus, we synthesized peptides of 10 that limited peptide solubility and lack of efficacy prevented residues each that progress from the N-terminus of ic2 to complete IC50 determination for all peptides (P24–27). The the C-terminus two amino acids at a time (Table 1). Beyond uM concentration ranges for activity of these peptides, and, the parent peptide, P11, this results in seven additional shallow concentration-effect relationships in, are typical for peptides (P21–P27). Agonist inhibition [59, 60]was used other peptides we and others have analyzed [48, 53]. as a measure of coupling efficacy. Any agent or process that Figure 2 gives results for the eight peptides’ ability to uncouples a receptor from its G-protein partner increases the foster incorporation of GTP into Gi using a radioactively probability that the receptor will be in a lower affinity state labeled, reasonably stable form of GTP ([35S] gamma-S- for agonist binding. This results in concentration-dependent GTP). Relative to control (buffer alone; no agonist nor agonist dissociation relationships that reflect affinity of the peptide) midloop residues as represented by peptide P23 are uncoupler for the G protein (and potentially by analogy most effective in directing incorporation of GTP into Gi. the affinity of the cognate receptor loop region for the G Efficacy declines in both N- and C-terminal directions from protein). Two determinants of G-protein activation (stable P23 although the results for P21 are anomalous in this regard. GTP binding to Gi and changes in intracellular cAMP It is not clear whether this result for P21 is meaningful or due concentration) were used to monitor a peptide’s ability to to experimental error although the results for intracellular Specific [ H]-8-OH-DPAT Control (Sp.γ [ S]-GTP incorp.)(%) binding (%) 4 International Journal of Peptides Table 2: ic2 Peptide mimic effect on [3H]8-OH-DPAT binding. All binding inhibition values are percent of control agonist (ag.) bound. The upper portion of the table is for peptides nearer the C-terminus, including P11 from Thiagaraj et al., 2007 [53]. These peptides decreased the specific high affinity binding of 5HT1aR agonist [ H]-8-OH-DPAT by 50% at the given concentration. The lower portion of the table (P24 on)isthe ic2peptidestowardthe Cterminus. Thesepeptideswerelesseffective at decreasing specific high affinity binding of [ H]-8- OH-DPAT, and values given are percent of control at the given concentration. Values for intracellular cAMP are relative to FSK-stimulated control. All values for incorporation of γ-[ S]-GTP into Gi are percent of control. Nominal values for control binding were 400 fmoles/mg protein. Peptide Conc. (uM) % cont. ag, bound, (SEM) [cAMP] (SEM) GTP Incorp. (SEM) Control 100 (6) 100 (7) 5HT 21 (4) 168 (12) P11 7 50 (1) 87 (8) 100 (3) P21 15 52 (4) 122 (8) 158 (11) P22 16 51 (2) 71 (2) 128 (9) P23 30 50 (22) 42 (4) 188 (10) P24 10 94 (9) 45 (7) 146 (17) P25 30 87 (12) 64 (3) 126 (10) P26 30 75 (19) 100 (5) 111 (9) P27 30 95 (5) 132 (6) 130 (7) Peptide effect on forskolin (FSK) stimulated cAMP γ -[ S]-GTP incorporation for P12 and P13 production 261 ± 9 204 ± 15 185.5 ± 9 20 150 124 ± 6 112 ± 6 100 ± 4 FSK 5HT P21 P22 P23 P24 P25 P26 P27 Figure 3: ic2 peptide effect on forskolin-stimulated cAMP pro- duction, a measure of activated-G-protein regulation of adenylyl cyclase. Forskolin (FSK) stimulated cAMP production by adenylyl cyclase (AC) is in CHO cells expressing the human 5HT1aR. FSK (30 uM) is the control, which is set to 100%. All other treatments are Control 5HT (10- P12 (3∗ 5HT/P12 P13 (5∗ 5HT/P13 expressed as a percent of the control value. Peptide concentrations 6 M) 10-5 M) 10-3 M) are 30 uM. All treatments include isobutylmethylxanthine (IBMX) an inhibitor of the metabolism of cAMP by phosphodiesterase. Figure 4: P12 and P13-stimulated incorporation of γ-[ S]-GTP ErrorisexpressedasSEM. control is the basal amount of γ-[ S]-GTP incorporated into Gi in CHO cells expressing the human 5HT1aR set as 100%. The Y axis is the percent of specifically bound γ-[ S]-GTP. All other treatments are percents of the control value. Peptide concentrations are 30 uM. cAMP (Figure 3) may shed some light on this situation. Note ∗ C P< 0.01 P12 versus control; P< 0.01 5HT versus 5HT/P12. that GTP binding by Gi is an agonist-dependent process P13 versus control P< 0.01; 5HT versus 5HT/P13 P< 0.01. (see 5HT in the Figure); thus, when peptides increase GTP incorporation above control level, the implication is that the peptides are representing native loop regions under the on both sides are parallel for GTP incorporation (Figure 2) influence of agonist. and cAMP concentrations (Figure 3). Note that basal levels Figure 3 shows a parallel set of results whereby the of intracellular cAMP are quite low, and the experimental peptides’ ability to change intracellular cAMP concentrations protocol for these experiments involves artificially raising following coupling to Gi is determined (control is the FSK cAMP concentrations via stimulation of adenylyl cyclase stimulated level; agonist; e.g., 5HT activates Gi and lowers with forskolin (control) and comparison of peptide results cAMP levels below the control reading). Again, peptide P23, to that produced by the agonist serotonin. representing mid-loop residues, is most effective. In contrast to the results for GTP incorporation in Figure 2, the cAMP results have a smooth drop off in efficacy on both sides 3.2. Intracellular Loop 3 (ic3). H5HT1aR’s ic3 is much larger of P23. Overall, the trends peaking at P23 and declining than ic2 (about 130 amino acids); nevertheless, we did a very FSK Stimulated cAMP (%) Control (Sp.γ [ S]-GTP incorp) (%) International Journal of Peptides 5 Table 3: ic3 Peptide mimic coupling and signal transduction data. 124 ± 7 Summary of data generated for all ic3 experiments with P12 and 100 ± 5 100 ± 6 P13. P1 is included as a reference, from Hayataka et al., 1998 [49]. Nominal values for control agonist binding were 400 fmoles/mg 60 ± 4 75 protein. 41 ± 1 50 ∗ [35S]-γ-S-GTP % Inhibition of Agonist (%) Peptide incorporation% FSK-stimulated 24 ± 1 inhibition above conro cAMP P1 50 (3 uM) 30 (1 uM) 10 (10 uM) FSK 5HT P12 P13 5HT/P12 5HT/P13 P12 28 (30 uM) 24 (30 uM) 0 (30 uM) Treatment P13 50 (15 uM) 12 (30 uM) −24 (30 uM) Figure 5: P12 and P13 effect of forskolin-stimulated cAMP pro- duction forskolin (FSK) stimulated cAMP production by adenylyl cyclase (AC) in CHO cells expressing the human 5HT1aR. These current investigation we have presented further information experiments were a measure of second messenger regulation by about the H5HT1aR/Gi interface that should provide testable G protein. FSK is the control, which is set to 100%. All other treatments are expressed as a percent of the control value. Peptide hypotheses anticipating the ultimate structural analysis of concentrations are 30 uM. All treatments include isobutylmethyl the receptor. xanthine (IBMX), an inhibitor of the metabolism of cAMP. 5HT Data collected in previous and current experiments versus 5HT/P12 and 5HT versus 5HT/P13 P< 0.05. have implicated a role for ic2 in receptor coupling and G- protein activation. The N-terminus end of ic2, involving the sequence IALDRYWAITDPIDYV and including pep- limited number of comparisons at the N-terminal (TM5) tides P11 (previous work) and P21–P23 (current work), is region of ic3, continuing preliminary work [50–53]and important for coupling to the G protein. Evidence for this using the same approach as with ic2 by synthetically building includes presence of the highly conserved DRY sequence 10-MER’s two amino acids at a time from the parent (P1; for GPCR’s [51] and from the present study, IC50’s for the Table 1). Table 3 gives coupling and activation summaries peptides’ coupling capacity, with ranges from 7 to 30 uM. for the two peptides, P12 and P13 (Table 1). As with the Decay of G-protein coupling activity was observed as the ic2 peptides, the ic3 peptides, P12 and P13, give shallow, peptides progress towards the C-terminus of ic2 (P24– uM concentration-effect relationships (data not shown 27). As the amino acids seem to wane in importance for in graphical form as in Figure 1). For coupling, if 50% receptor coupling, they increase for part of the distance in is listed, then that is the IC 50; if another value is listed, importance for G-protein activation with its peak at the that is the maximum inhibition possible with the highest P23 amino acid stretch WAITDPIDYV. This is clearly shown soluble concentration. Both P12 and P13 produce small by the bell-shaped progression of the data bars for the but significant incorporation increases of GTP based upon incorporation of γ-[ S]-GTP into Gi (Figure 2). This can the amount of [ S]-GTP incorporated into Gi (Figure 4), be superimposed over the inverted bell-shaped depression while the outcomes for changes in intracellular cAMP for intracellular levels of FSK-stimulated cAMP production concentrations are more complex (Figure 5): for peptide P12, (Figure 3) following peptide treatment. intracellular cAMP concentration is not altered; unusually, The C-terminal end of ic2 consisting of the amino peptide P13 actually increases intracellular cAMP concen- acids RTPRPR may serve as an anchor, helping to hold the tration. A possible explanation for the combined results for amino terminal of ic2 in a favorable orientation for coupling P12 and P13 is given in the Discussion section. to the G protein [48]. Also interesting about the carboxy terminal end of ic2 is the presence of the 2 proline (P) residues separated by only 1 amino acid. These proline 4. Discussion residues in close proximity to each other introduce a kink H5HT1aR is linked to numerous important physiological in the receptor structure constraining its range of motion. and pathological processes. Additionally, the receptor is a These data demonstrates the clear role for H5HT1aR’s ic2 in close relative, not only of other 5HT1 type receptors, but coupling receptor to G protein, and toward the loop’s middle, also the beta adrenergic receptors and other GPCR’s [13, 42, G-protein activation. 55, 56]. Because of these characteristics, structural determi- For ic3, the inhibition of AC by Gi is an important nations of the receptor are crucial matters. Despite recent regulator of intracellular signal transduction. The current critical structural advances with the beta adrenergic receptors peptides tested, P12 and P13, differed in their ability to [10, 15, 16, 18], the 5HT1aR is uncrystallized and its struc- regulate this step in the cascade. P12 was unable to decrease ture awaits X-ray analysis [22]. the FSK-stimulated levels of cAMP (Figure 5). This is in In previous work [49, 50, 53, 54], we have demonstrated contrast to the action of 5HT which was able to significantly the utility of an agonist-based inhibition system associated decrease the FSK-stimulated levels of cAMP. P13 had the with signal transduction parameters to study interactions opposite effect; it increased cAMP concentrations (Figure 5)! of the receptor with its cognate G protein, Gi. In the This suggests that the two new amino acids (AD added to FSK stimulated cAMP (%) 6 International Journal of Peptides form P13) from ic3 are potentially at the beginning of a efficacious tertiary structure without the full loop being region of the loop which has negative regulatory properties present. It is crucial to point out that the parent ic3 peptide on Gi blunting its normal ability to regulate AC. It is (P1) contains the full TVKK sequence at its N-terminus. interesting to speculate about the differences in data from This sequence is part of the so-called Ric-8 [62, 63]region the γ-[ S]-GTP (Figure 4) incorporation assays and cAMP that has been shown in other GPCR as crucial to G-protein assays (Figure 5). P12 slightly increases GTP incorporation regulation. Significantly, the P1 relatives (P12 and P13) while P13 statistically does not. Thus, P12 activates Gi but under discussion in this communication are at a transition cAMP changes do not ensue. P13 does not activate Gi, point for this sequence; P12 contains the full TVKK stretch but a cAMP change occurs in the atypical direction. With while P13 has lost the T! One additional observation may the relatively small changes produced by these two peptides be pertinent. For GTP incorporation, for both peptides, the in both signaling measures, one possibility is experimental combination of peptide plus 5HT produces markedly greater error that has not been accounted for. It is possible that the incorporation than that produced by 5HT alone. This peptides are acting at some sites other than the proposed may suggest that 5HT and the peptides may be perturbing receptor-G-protein interface or that the process at the inter- separate sites on the receptor and/or G protein. face is more complex. In summary, this peptide mimic study for intracellular The most tantalizing possibility is that the newly explored loops 2 and 3 of the H5HT1aR was designed to examine region represented by P12 and P13 is the beginning of a which segments were involved in coupling and activation region of ic3 involved in coupling of receptor to G protein of Gi. The results reported here in combination with still capable of regulating Gi. Additionally, the perturbation previously reported work conclude that the amino terminal of Gi in this case involves different conformational changes ends of ic2 and ic3 are important for coupling the receptor that activate Gi but in a novel way. This would produce and G protein. The activation of G-protein peaks at P23 the opposite effect on cAMP concentration and would be (WAITDPIDYV) in ic2 (mid-loop). The activity is decreased equivalent to the downstream actions of an inverse agonist at as the structures move in either direction away from this the ligand binding site. Since 5HT1aR is capable of constitu- core sequence. The curious results of increased cAMP tive activity [25], inverse agonism is possible, and it will be concentrations caused by P13 suggests that the two new fascinating to see if the P12/P13 region is involved in this amino acids (AD) in P13 are the beginning of a new region activity once the crystal structure is available. In this context of ic3 which has negative regulatory properties on Gi. That then, P12 would represent a transitional region between is, the new region may be one that is not normally activated “normal” and “atypical” Gi regulation while P13 is in the by agonists; however, in the presence of inverse agonists and atypical subregion. the different conformational changes they produce, the new While the data support this region’s (P12/P13) role in region may couple to and activate Gi in a way that regulates receptor-G-protein coupling, the peptides’ ability to uncou- AC in a way we define as inverse agonism. The combined ple declines relative to previously studied peptides whose results with H5HT1aR ic2 and ic3 peptides should lead structures represent segments closer to ic3’s N-terminus. to testable crystallographic hypotheses with drugs having P12 and 13 are beyond (toward the C-terminus) the key differential intrinsic activities. Beyond the final judgment of RFRI region of P1 previously identified as key to that these peptide probes in the structural sense, the information part of 5HT1aR’s ic3-N-terminus responsible for G-protein produced may be useful as independent pharmacological activation [50]. observations. Pragmatic implications of the work may be Varrault et al. [48] demonstrated that the C-terminal relevant in a framework where the multiple, differential section of i3 is involved in G protein coupling and regulation. activities of the peptides can be used by medicinal chemists So, if our work can be interpreted to mean that peak coupling to build unique pharmacological agents targeting unutilized and activating properties are associated with ic3’s N-terminal sites at the receptor-G-protein interface. residues and Varrault’s work can be interpreted to mean that peak coupling and activating properties are associated with ic3’s C-terminal residues, then what role will hold for the vast Acknowledgments internal region of the loop in 5HT1aR? GPCR ic3’s are vari- able in size in rhodopsin versus 5HT1aR and BAR’s, which The authors would like to express deepest appreciation to the have larger ic3 loops (at least twice the size of rhodopsin’s Department of Biomedical and Pharmaceutical Sciences, the ic3). It would be meaningful to extend this peptide approach Skaggs School of Pharmacy, the College of Health Professions into the midloop region of H5HT1a’s ic3, and then as a crys- and Biomedical Sciences, all of The University of Montana tal structure becomes available the comparisons of 5HT1aR (UM), Missoula. Without the resources and human support loop function with BAR and rhodopsin will be fascinating. of these units and The University, the project could not Neither of the peptides (P12 and P13) are as potent have occurred. Special thanks to Dr. David Freeman for as 5HT at incorporating GTP into Gi . It is possible that his editorial assistance. Research was conducted under the multiple regions are responsible for G-protein activation, generous and essential sponsorship of the following NIH and the individual peptides mimic only part of this structure Grants: UM-CHPBS Endowment Fund Program NIH S21- [61], thereby producing a diminished effect relative to 5HT. MD000236, RR10169 and GM/OD 54302-01/02, and P20 Also, a given peptide region, even one that is absolutely RR 15583 to the UM COBRE Center for Structural and critical in the native structure, may not have the most Functional Neurosciences from NCRR. International Journal of Peptides 7 References [19] V. P. Jaakola, M. T. Griffith,M.A.Hansonetal., “The 2.6 angstrom crystal structure of a human A2A adenosine recep- [1] E. C. Azmitia, “Serotonin and brain: evolution, neuroplastic- tor bound to an antagonist,” Science, vol. 322, no. 5905, pp. ity, and homeostasis,” International Review of Neurobiology, 1211–1217, 2008. vol. 77, pp. 31–56, 2006. [20] B. Wu, E. Y. T. Chien, C. D. Mol et al., “Structures of the [2] M.Filip andM.Bader,“Overview on 5-HT receptorsand CXCR4 chemokine GPCR with small-molecule and cyclic their role in physiology and pathology of the central nervous peptide antagonists,” Science, vol. 330, no. 6007, pp. 1066– system,” Pharmacological Reports, vol. 61, no. 5, pp. 761–777, 1071, 2010. 2009. [21] E. Y. T. Chien, W. Liu, Q. Zhao et al., “Structure of the human dopamine D3 receptor in complex with a D2/D3 selective [3] D. E. Nichols and C. D. Nichols, “Serotonin receptors,” Chem- antagonist,” Science, vol. 330, no. 6007, pp. 1091–1095, 2010. ical Reviews, vol. 108, no. 5, pp. 1614–1641, 2008. [22] S. Topiol and M. Sabio, “X-ray structure breakthroughs in the [4] N. M. Barnes and T. Sharp, “A review of central 5-HT receptors GPCR transmembrane region,” Biochemical Pharmacology, and their function,” Neuropharmacology,vol. 38, no.8,pp. vol. 78, no. 1, pp. 11–20, 2009. 1083–1152, 1999. [23] J. R. Raymond, Y. V. Mukhin, T. W. Gettys, and M. N. Gar- [5] D. Hoyer, D. E. Clarke, J. R. Fozard et al., “International novskaya, “The recombinant 5-HT(1A) receptor: G protein Union of Pharmacology classification of receptors for 5-hy- coupling and signalling pathways,” British Journal of Pharma- droxytryptamine (serotonin),” Pharmacological Reviews, vol. cology, vol. 127, no. 8, pp. 1751–1764, 1999. 46, no. 2, pp. 157–203, 1994. [24] J. R. Raymond, Y. V. Mukhin, A. Gelasco et al., “Multiplicity [6] P. J. Goadsby, A. R. Charbit, A. P. Andreou, S. Akerman, and of mechanisms of serotonin receptor signal transduction,” P. R. Holland, “Neurobiology of migraine,” Neuroscience, vol. Pharmacology and Therapeutics, vol. 92, no. 2-3, pp. 179–212, 161, no. 2, pp. 327–341, 2009. [7] K. K. Parker, “Involvement of adipokines in migraine head- [25] J. C. Martel,A.M.Ormiere,N.Leduc,M.B.Assie, D. Cussac, ache,” in Extracellular & Intracellular Signaling,J.D.Adams and A. Newman-Tancredi, “Native rat hippocampal 5-HT 1A and K. K. Parker, Eds., Royal Society of Chemistry, Cambridge, receptors show constitutive activity,” Molecular Pharmacology, UK, 2011. vol. 71, no. 3, pp. 638–643, 2007. [26] J. Guptarak, A. Selvamani, and L. Uphouse, “GABAA-5-HT [8] H. R. Bourne, “G-proteins and GPCRs: from the beginning,” 1A receptor interaction in the mediobasal hypothalamus,” Brain Ernst Schering Foundation symposium proceedings,no. 2, pp. Research, vol. 1027, no. 1-2, pp. 144–150, 2004. 1–21, 2006. [27] E. B. Russo, A. Burnett, B. Hall, and K. K. Parker, “Agonistic [9] S. C. Sinha and S. R. Sprang, “Structures, mechanism, regula- properties of cannabidiol at 5-HT receptors,” Neurochemical 1A tion and evolution of class III nucleotidyl cyclases,” Reviews of Research, vol. 30, no. 8, pp. 1037–1043, 2005. Physiology, Biochemistry and Pharmacology, vol. 157, pp. 105– [28] C. P. Muller,R.J.Carey,J.P.Huston, andM.A.deSouza Silva, 140, 2006. “Serotonin and psychostimulant addiction: focus on 5-HT - 1A [10] B. K. Kobilka, “G protein coupled receptor structure and receptors,” Progress in Neurobiology, vol. 81, no. 3, pp. 133– activation,” Biochimica et Biophysica Acta, vol. 1768, no. 4, pp. 178, 2007. 794–807, 2007. [29] C. Jonnakuty and C. Gragnoli, “What do we know about [11] B. K. Kobilka and X. Deupi, “Conformational complexity of serotonin?” Journal of Cellular Physiology, vol. 217, no. 2, pp. G-protein-coupled receptors,” Trends in Pharmacological Sci- 301–306, 2008. ences, vol. 28, no. 8, pp. 397–406, 2007. [30] D. Kozaric-Kovacic, “Psychopharmacotherapy of posttrau- [12] R. J. Lefkowitz, J. P. Sun, and A. K. Shukla, “A crystal clear matic stress disorder,” Croatian Medical Journal, vol. 49, no. view of the β -adrenergic receptor,” Nature Biotechnology, vol. 4, pp. 459–475, 2008. 26, no. 2, pp. 189–191, 2008. [31] J. L. Rausch, M. E. Johnson, K. E. Kasik, and S. M. Stahl, “Tem- perature regulation in depression: functional 5HT1A receptor [13] D. M. Rosenbaum, S. G. F. Rasmussen, and B. K. Kobilka, adaptation differentiates antidepressant response,” Neuropsy- “The structure and function of G-protein-coupled receptors,” chopharmacology, vol. 31, no. 10, pp. 2274–2280, 2006. Nature, vol. 459, no. 7245, pp. 356–363, 2009. [32] E. Akimova, R. Lanzenberger, and S. Kasper, “The serotonin- [14] J. M. Baldwin, “Structure and function of receptors coupled to 1A receptor in anxiety disorders,” Biological Psychiatry, vol. 66, G proteins,” Current Opinion in Cell Biology,vol. 6, no.2,pp. no. 7, pp. 627–635, 2009. 180–190, 1994. [33] G. S. Kranz, S. Kasper, and R. Lanzenberger, “Reward and the [15] V. Cherezov,D.M.Rosenbaum,M.A.Hansonetal., “High- serotonergic system,” Neuroscience, vol. 166, no. 4, pp. 1023– -adren- resolution crystal structure of an engineered human β 1035, 2010. ergic G protein-coupled receptor,” Science, vol. 318, no. 5854, [34] L. A. Catapano and H. K. Manji, “G protein-coupled receptors pp. 1258–1265, 2007. in major psychiatric disorders,” Biochimica et Biophysica Acta, [16] S. G. F. Rasmussen, H. J. Choi, D. M. Rosenbaum et al., “Crys- vol. 1768, no. 4, pp. 976–993, 2007. tal structure of the human β adrenergic G-protein-coupled [35] B. le Francois, M. Czesak, D. Steubl, and P. R. Albert, “Tran- receptor,” Nature, vol. 450, no. 7168, pp. 383–387, 2007. scriptional regulation at a HTR1A polymorphism associated [17] D. M. Rosenbaum, V. Cherezov, M. A. Hanson et al., “GPCR with mental illness,” Neuropharmacology,vol. 55, no.6,pp. engineering yields high-resolution structural insights into β - 2 977–985, 2008. adrenergic receptor function,” Science, vol. 318, no. 5854, pp. [36] L. B. Resstel,R.F.Tavares,S.F.Lisboa, S. R. Joca,F.M.Correa, 1266–1273, 2007. and F. S. Guimaraes, “5-HT receptors are involved in the 1A [18] D. M. Rosenbaum, C. Zhang, J. A. Lyons et al., “Structure and cannabidiol-induced attenuation of behavioral & cardiovascu- lar responses to acute restraint stress in rats,” British Journal of function of an irreversible agonist-β adrenoceptor complex,” Nature, vol. 469, no. 7329, pp. 236–240, 2011. Pharmacology, vol. 156, no. 1, pp. 181–188, 2009. 8 International Journal of Peptides [37] J. Savitz, I. Lucki, and W. C. Drevets, “5-HT receptor func- [53] H. V. Thiagaraj, T. C. Ortiz, M. C. Devereaux Jr., B. Seaver, B. 1A tion in major depressive disorder,” Progress in Neurobiology, Hall, and K. K. Parker, “Regulation of G proteins by human vol. 88, no. 1, pp. 17–31, 2009. 5-HT receptor TM3/i2 and TM5/i3 loop peptides,” Neuro- 1A chemistry International, vol. 50, no. 1, pp. 109–118, 2007. [38] G. V. Carr and I. Lucki, “The role of serotonin receptor sub- types in treating depression: a review of animal studies,” Psy- [54] B. Hall, A. Burnett, A. Christians et al., “Thermodynamics of chopharmacology, vol. 213, no. 2-3, pp. 265–287, 2011. peptide and non-peptide interactions with the human 5HT 1A receptor,” Pharmacology, vol. 86, no. 1, pp. 6–14, 2010. [39] P. N. Yadav, A. I. Abbas, M. S. Farrell et al., “The presynaptic component of the serotonergic system is required for cloza- [55] A. Fargin, J. R. Raymond, M. J. Lohse, B. K. Kobilka, M. G. pine’s efficacy,” Neuropsychopharmacology,vol. 36, no.3,pp. Caron, and R. J. Lefkowitz, “The genomic clone G-21 which 638–651, 2011. resembles a β-adrenergic receptor sequence encodes the 5- HT receptor,” Nature, vol. 335, no. 6188, pp. 358–360, 1988. [40] J. P. Changeux and S. J. Edelstein, “Allosteric mechanisms of 1A signal transduction,” Science, vol. 308, no. 5727, pp. 1424– [56] B. K. Kobilka, T. Frielle, S. Collins et al., “An intronless gene 1428, 2005. encoding a potential member of the family of receptors cou- pled to guanine nucleotide regulatory proteins,” Nature, vol. [41] J. H. Turner, M. N. Garnovskaya, and J. R. Raymond, “Sero- 329, no. 6134, pp. 75–79, 1987. tonin 5-HT receptor stimulates c-Jun N-terminal kinase 1A and induces apoptosis in Chinese hamster ovary fibroblasts,” [57] M. M. Bradford, “A rapid and sensitive method for the quan- Biochimica et Biophysica Acta, vol. 1773, no. 3, pp. 391–399, titation of microgram quantities of protein utilizing the prin- 2007. ciple of protein dye binding,” Analytical Biochemistry, vol. 72, no. 1-2, pp. 248–254, 1976. [42] M. C. Lagerstrom and H. B. Schioth, “Structural diversity of G protein-coupled receptors and significance for drug discov- [58] T. Wieland and K. H. Jakobs, “[1] Measurement of receptor- ery,” Nature reviews. Drug Discovery, vol. 7, no. 4, pp. 339–357, stimulated guanosine 5 -O-(γ-thio)triphosphate binding by G 2008. proteins,” Methods in Enzymology, vol. 237, pp. 3–13, 1994. [43] M. J. Millan, P. Marin, J. Bockaert, and C. Mannoury la Cour, [59] M. E. Maguire, P. M. van Arsdale, and A. G. Gilman, “An “Signaling at G-protein-coupled serotonin receptors: recent agonist specific effect of guanine nucleotides on binding to the advances and future research directions,” Trends in Pharma- β adrenergic receptor,” Molecular Pharmacology, vol. 12, no. 2, cological Sciences, vol. 29, no. 9, pp. 454–464, 2008. pp. 335–339, 1976. [44] B. Sjgren,L.L.Blazer, andR.R.Neubig, “Regulatorsof [60] S. J. Peroutka, R. M. Lebovitz, and S. H. Snyder, “Sero- G protein signaling proteins as targets for drug discovery,” tonin receptor binding sites affected differentially by guanine Progress in Molecular Biology and Translational Science, vol. 91, nucleotides,” Molecular Pharmacology, vol. 16, no. 3, pp. 700– no. C, pp. 81–119, 2010. 708, 1979. [45] J. A. Allen and B. L. Roth, “Strategies to discover unexpected [61] S. Kalipatnapu and A. Chattopadhyay, “Membrane organiza- targets for drugs active at G protein-coupled receptors,” An- tion and function of the serotonin receptor,” Cellular and 1A nual Review of Pharmacology and Toxicology, vol. 51, pp. 117– Molecular Neurobiology, vol. 27, no. 8, pp. 1097–1116, 2007. 144, 2011. [62] G. G. Tall, A. M. Krumins, and A. G. Gilman, “Mammalian [46] S. Ganguly, A. H. A. Clayton, and A. Chattopadhyay, “Orga- Ric-8A (synembryn) is a heterotrimeric Gα protein guanine nization of higher-order oligomers of the serotonin receptor nucleotide exchange factor,” The Journal of Biological Chem- 1A explored utilizing homo-FRET in live cells,” Biophysical Jour- istry, vol. 278, no. 10, pp. 8356–8362, 2003. nal, vol. 100, no. 2, pp. 361–368, 2011. [63] C. J. Thomas, G. G. Tall, A. Adhikari, and S. R. Sprang, “Ric- [47] A. Ivetac and J. Andrew McCammon, “Mapping the druggable 8A catalyzes guanine nucleotide exchange on Gαi1 bound to allosteric space of g-protein coupled receptors: a fragment- the GPR/GoLoco exchange inhibitor AGS3,” The Journal of based molecular dynamics approach,” Chemical Biology and Biological Chemistry, vol. 283, no. 34, pp. 23150–23160, 2008. Drug Design, vol. 76, no. 3, pp. 201–217, 2010. [48] A. Varrault, Dung Le Nguyen, S. McClue, B. Harris, P. Jouin, and J. Bockaert, “5-Hydroxytryptamine receptor synthetic 1A peptides: mechanisms of adenylyl cyclase inhibition,” The Journal of Biological Chemistry, vol. 269, no. 24, pp. 16720– 16725, 1994. [49] K. Hayataka, M. F. O’Connor, N. Kinzler, J. T. Weber, and K. K. Parker, “A bioactive peptide from the transmembrane 5-intracellular loop 3 region of the human 5HT receptor,” 1A Biochemistry and Cell Biology, vol. 76, no. 4, pp. 657–660, 1998. [50] T. C. Ortiz, M. C. Devereaux, and K. K. Parker, “Structural variants of a human 5-HT receptor intracellular loop 3 1A peptide,” Pharmacology, vol. 60, no. 4, pp. 195–202, 2000. [51] N. Kushwaha, S. C. Harwood, A. M. Wilson et al., “Molecular determinants in the second intracellular loop of the 5-hy- droxytryptamine-1A receptor for G-protein coupling,” Molec- ular Pharmacology, vol. 69, no. 5, pp. 1518–1526, 2006. [52] A. O. Shpakov and M. N. Pertseva, “Molecular mechanisms for the effect of mastoparan on G proteins in tissues of verte- brates and invertebrates,” Bulletin of Experimental Biology and Medicine, vol. 141, no. 3, pp. 302–306, 2006. 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Intracellular Loop 2 Peptides of the Human 5HT1a Receptor are Differential Activators of Gi

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Copyright © 2012 Brian Hall et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Hindawi Publishing Corporation International Journal of Peptides Volume 2012, Article ID 490734, 8 pages doi:10.1155/2012/490734 Research Article Intracellular Loop 2 Peptides of the Human 5HT1a Receptor are Differential Activators of Gi Brian Hall, Carley Squires, and Keith K. Parker Department of Biomedical and Pharmaceutical Sciences (MPH I02), Center for Structural and Functional Neuroscience, Skaggs School of Pharmacy, The University of Montana, 32 Campus Drive No. 1552, Missoula, MT 59812-1552, USA Correspondence should be addressed to Keith K. Parker, keith.parker@umontana.edu Received 27 December 2011; Revised 10 February 2012; Accepted 24 February 2012 Academic Editor: Piero Andrea Temussi Copyright © 2012 Brian Hall et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Peptide mimics of intracellular loop 2 (ic2) of the human 5HT1a receptor have been studied with respect to their ability to inhibit agonist binding via interference with receptor-G-protein coupling. These peptides give shallow concentration-effect relationships. Additionally, these peptides have been studied with respect to their ability to trigger the signal transduction system of this Gi-coupled receptor. Two signaling parameters have been quantified: concentration of intracellular cAMP and changes in incorporation into the G protein of a stable analog of GTP. In both cases, peptide mimics near midloop of ic2 actually show agonist activity with efficacy falling off toward both loop termini near TM 3 and TM 4. Previous results have suggested that the loop region near the TM3/ic2 interface is primarily responsible for receptor-G-protein coupling, while the current result emphasizes the mid- ic2 loop region’s ability to activate the G protein following initial coupling. A limited number of peptides from the receptor’s TM5/ic3 loop vicinity were also studied regarding agonist inhibition and G-protein activation. These peptides provide additional evidence that the human 5HT1a receptor, TM5/ic3 loop region, is involved in both coupling and activation actions. Overall, these results provide further information about potential pharmacological intervention and drug development with respect to the human 5HT1a receptor/G-protein system. Finally, the structural evidence generated here provides testable models pending crystallization and X-ray analysis of the receptor. 1. Introduction greater fundamental knowledge but to therapeutic develop- ment. Regulation of serotonergic (5-hydroxytryptamine; 5HT) Various receptors (R), including the 5HT3R’s that are function in animals impacts numerous physiological and ligand-controlled ion channels, are crucial to these regula- pathological processes [1]. 5HTisbroadly representedinbi- tory processes [4]. All other known 5HTR’s are structurally ological systems as a regulator and modulator via nervous, different than these ion channels, being serpentine mem- hormonal, and autacoidal means [2–5]. For example, sero- brane R’s [8], coupled (C) to the cells interior by G (GTP tonin [6] has been implicated in the pathophysiology of binding) proteins (P), which in turn regulate key effectors migraine. This association with migraine is shared with many such as adenylyl cyclase (AC, [9]). These GPCR’s share the other factors including adipokines such as leptin; hypothala- structural characteristic of 7-transmembrane (7TM) helical mic hormones, Orexin A and B (also known appetite regula- segments [10–13]. For many years, the only crystal structure tors as is 5HT); numerous neurotransmitters [7]; autacoids; was of rhodopsin, the prototype GPCR, in its interaction hormones, and ions like calcium, and magnesium. The with the G-protein transducin [14]. Recently a breakthrough range of biological molecules that interact with serotonergic has occurred, with crystallization of the beta-adrenergic processes suggests that various signaling pathways may be receptor (BAR) and publication of X-ray structures [15– shared, and that the potential for dynamic, collaborative 18]. This long-awaited event has set the stage for other regulation exists. Better understanding of the molecular basis GPCR. Progressive developments have been demonstrated by underlying these signaling processes is not only critical to crystal structures for the adenosine A2R [19], the CXDR4 2 International Journal of Peptides chemokine R [20], and the dopamine D3R [21]. Crystal Reactions were stopped by addition of 4 mL of ice-cold structures for other GPCR, including that those 5HTR’s that 50 mM Tris buffer, pH 7.4, and vacuum filtration on glass are GPCR’s should soon follow [22]. fiber filters (Whatman GF/B). Filters were rinsed twice in Of the GPCR recognized as 5HTR, the 5HT1aR (a relative 5 mL of ice-cold Tris buffer, dried, and counted in 5 mL of of BAR) is one of the most highly studied [23–25], and Ecoscint (National Diagnostics) liquid scintillation fluid in a it has been associated with physiological and pathological Beckman LS 6500. Homogenates were assayed for protein to processes as diverse as thermoregulation, cognitive flexibility, maintain a nominal value of 50 μg protein per filter [57]. All and control of mood [26–33]. Depression, underlying anxi- tubes were run in triplicate. ety disorders, and related psychopathologies are a particular theme [34–39]. Multiple strategies have been used to dissect 2.4. cAMP Assay. CHO cells were cultured to confluency in the complex pathways underscoring these physiological 12- or 24-well plates. Medium was aspirated, and the cells and pathological processes [40–47]. One approach centers were rinsed twice in warm, serum-free F-12 medium. Cells around analysis of allosteric sites of action on receptors. were incubated for 20 min. at 37 Cin0.5mlsofserum-free Peptide mimics of intracellular loop regions of 5HT1aR have F-12 medium containing 100 uM isobutylmethylxanthine been used as probes of the receptor-G-protein interface in (IBMX) and the following substances (final concs.) alone or this context [48–54]. The current communication continues in combination (see Figures 3 and 5): 30 μM forskolin (FSK; our analysis with these peptide probes particularly emphasiz- for all treatments); 1 μM 5-HT; peptide concentrations as ing intracellular loop 2 (ic2) with some, limited comparative noted in figure legends. Reactions were stopped by aspiration data from intracellular loop 3 (ic3). The results with ic2 of medium and addition of 0.5 mL of 100 mM HCl. After and ic3 are suggestive of potential sites for regulation and 10 min., well contents were removed and centrifuged at therapeutic drug development. 4000 rpm. Supernatants were diluted in 100 mM HCl, and cAMP was quantified [53] directly in a microplate format by enzyme immunoassay (EIA) with a kit from Assay Designs 2. Materials and Methods (Ann Arbor). Triplicate-independent samples were assayed. 2.1. Cell Culture. Chinese Hamster ovary (CHO) cells ex- 2.5. [35S]GTPγS Assay. H5-HT1aR membranes from trans- pressing the H5-HT1aR [55, 56]wereculturedinHam’s F-12 fected CHO cells were incubated with 5-HT (0.1 μM) and/or medium fortified with 10% fetal calf serum and 200 ug/mL peptide concentrations as noted in figure legends (see geneticin. Cultures were maintained at 37 C in a humidified Figures 2 and 4) and the following incubation mixture: atmosphere of 5% CO . Cells were subcultured or assayed 20 mM HEPES buffer, pH 7.4, 5 mM MgCl ,1mM EDTA, upon confluency (5–8 days). Cloned H5-HT1aR was kindly 2 1 mM DTT, 100 mM NaCl, 100 uM GDP, 10 μM pargyline, provided by Dr. John Raymond (Medical U. of South 0.2 mM ascorbate, and 0.1 nM [35S]GTPγS[53, 58]. Mix- Carolina; [41]). The cell line has been tested for mycoplasma tures were incubated for 30 min. at 30 C, and were terminat- with a PCR kit (ATCC) and is free of contamination. ed by dilution in cold buffer. The mixture was filtered on GF/C filters, rinsed twice in buffer, dried and counted by 2.2. Receptor Preparation. Cells were trypsinized and cen- liquid scin-tillation. All values reported in are for specific trifuged at low speed in ice-cold medium [53]. The pellet binding (total nonspecific) of triplicates. Nonspecific bind- was resuspended in ice-cold Earle’s Balanced Salt Solution ing was determined in the presence of cold γ-S-GTP (10 uM). followed by centrifugation. Cells were resuspended in 10 mL Negative controlisthe abovemixture minustestdrugor of ice-cold binding buffer (50mM Tris, 4mM CaCl ,10 μM 5HT. Positive control contains 5HT. pargyline, and pH 7.4), homogenized with Teflon-glass, and centrifuged for 450,000 g-min. at 4 C. For a crude membrane 2.6. Data Analysis. All statistics (means, standard errors of preparation, the pellet was resuspended in 30 mL of ice-cold the mean (SEM), t tests and ANOVA, Pearson correlation binding buffer, homogenized on Teflon-glass and then by coefficients (r), and graphical procedures (including drug- Polytron (setting 4) for 5 seconds, and stored on ice and receptor-binding analysis) were conducted with PSI-Plot assayed within the next 1.5 hours [54]. (Version 8) software (Poly Software International), Prism (version 4.0c), or using a Hewlett-Packard Graphing Calcu- lator, HP48. The apriori was α = 0.05 for all experiments. 2.3. Assay of Receptor Activity. Binding of the agonist [3H]8- Experiments were conducted with a minimum of n = 3, in OH-DPAT ([3H]8-hydroxy-2-(di-n-propylamino)tetralin) triplicate. Most experiments were n = 3–5. In some cases to H5-HT1aR followed well-characterized protocols (indicated in figure legends), different n’s and multiplicates [49, 50, 53]. Radioligands were purchased from New England were used. Nuclear (NEN), Boston, MA, and 1 mL reaction mixtures, in triplicate, were incubated for 30 min. in a 30 C shaker. The 1 mL mixture was 700 μLofreceptorpreparation; 2.7. Peptide Preparation. These highly purified (greater than 100 μL of binding buffer (for total binding) or 10 μM 5-HT 95%) peptides were purchased from New England Peptide (for nonspecific binding), 100 μL of the tritiated agent LLC. The peptides are segments of ic2 and ic3 of the cloned (concentration of 0.5 nM [3H] 8-OH-DPAT), and 100 μLof H5HT1aR. Peptides stored at −20 C were initially dissolved peptide or binding buffer in the case of controls. in deionized water. Subsequent dilutions were in binding International Journal of Peptides 3 Table 1: ic2 and ic3 peptide mimics. The primary amino acid seq- uences for the H5HT1aR ic2 loop peptide mimics P11 and P’s 21– 120 27, and for ic3 (P1, P12, and P13). The receptor’s amino terminal is to the left. Sequences for H5HT1aR from Kobilka et al., 1987 [56]. P11 is from a previous study by Thiagaraj et al., 2007 [53], and P1 from Hayataka et al., 1988 [49] (both included for comparative purposes). 0 5 10 15 20 25 30 35 P11 IALDRYWAITD P21 LDRYWAITDP P21 concentration μM P22 RYWAITDPID Figure 1: P21 noncentration-dependent displacement of bound 8- P23 WAITDPIDYV OH-DPAT. This curve represents the change in specific binding of P24 ITDPIDYVNK [ H]-8-OH-DPAT, a 5HT1aR agonist, to the receptor in the pres- ence of various concentrations of the ic2 peptide mimic P21. Nom- P25 DPIDYVNKRT inal binding of agonist at control levels was 400 fmoles/mg protein. P26 IDYVNKRTPR P27 YVNKRTPRPR P1 IFRAARFRIRKTVKK Peptide effect on γ-[ S]-GTP P12 KTVKKVEKTG incorporation P13 VKKVEKTGAD buffer. The peptides examined in this study are listed in Table 1. Control 5HT P21 P22 P23 P24 P25 P26 P27 3. Results Figure 2: ic2 peptide effect on γ-[ S]-GTP incorporated into Gi, 3.1. Intracellular Loop 2 (ic2). ThesizeofH5HT1a’sic2 a measure of G-protein activation. Control is the basal amount (about 20 amino acids) makes it a tempting target for analyz- 35 of γ-[ S]-GTP incorporated into Gi in CHO cells expressing ing the loop’s coupling to and activation of Gi [23, 24]. Our the human 5HT1aR, set as 100%. The Y-axisisthe percentof previous work with ic2 emphasized the N-terminal region of specifically bound (total minus nonspecific) γ-[ S]-GTP. All other the loop with a peptide we call P11 (Table 1). Results with treatments are percents of the control value. All peptides are 30 uM −7 concentration and 5HT 10 M concentration. Error is expressed as this peptide suggest that the loop residues near TM 3 are SEM. vital for coupling of the loop to Gi but are not involved in G- proteinactivation[53]. Results from the Varrault group [48] looked at the entire loop without distinguishing subregions; their conclusions were that the entire loop is responsible perturb G protein following coupling. The overall results for for activation (they did not differentiate between coupling these eight peptides are in Table 2. and activation). The following question arises: can coupling As shown in Figure 1 with results from peptide P21 as and activation characteristics be identified for the loop an example, these peptides give shallow concentration-effect on a subregional basis? Our preliminary work at the N- curves for the measure of coupling and agonist inhibition. terminal aspect of H5HT1a’a ic3 suggested to us that the Similar experiments with all peptides form the basis for techniques we use could be productive in addressing such a the summarized coupling results found in Table 2.Note question [49, 50, 53]. Thus, we synthesized peptides of 10 that limited peptide solubility and lack of efficacy prevented residues each that progress from the N-terminus of ic2 to complete IC50 determination for all peptides (P24–27). The the C-terminus two amino acids at a time (Table 1). Beyond uM concentration ranges for activity of these peptides, and, the parent peptide, P11, this results in seven additional shallow concentration-effect relationships in, are typical for peptides (P21–P27). Agonist inhibition [59, 60]was used other peptides we and others have analyzed [48, 53]. as a measure of coupling efficacy. Any agent or process that Figure 2 gives results for the eight peptides’ ability to uncouples a receptor from its G-protein partner increases the foster incorporation of GTP into Gi using a radioactively probability that the receptor will be in a lower affinity state labeled, reasonably stable form of GTP ([35S] gamma-S- for agonist binding. This results in concentration-dependent GTP). Relative to control (buffer alone; no agonist nor agonist dissociation relationships that reflect affinity of the peptide) midloop residues as represented by peptide P23 are uncoupler for the G protein (and potentially by analogy most effective in directing incorporation of GTP into Gi. the affinity of the cognate receptor loop region for the G Efficacy declines in both N- and C-terminal directions from protein). Two determinants of G-protein activation (stable P23 although the results for P21 are anomalous in this regard. GTP binding to Gi and changes in intracellular cAMP It is not clear whether this result for P21 is meaningful or due concentration) were used to monitor a peptide’s ability to to experimental error although the results for intracellular Specific [ H]-8-OH-DPAT Control (Sp.γ [ S]-GTP incorp.)(%) binding (%) 4 International Journal of Peptides Table 2: ic2 Peptide mimic effect on [3H]8-OH-DPAT binding. All binding inhibition values are percent of control agonist (ag.) bound. The upper portion of the table is for peptides nearer the C-terminus, including P11 from Thiagaraj et al., 2007 [53]. These peptides decreased the specific high affinity binding of 5HT1aR agonist [ H]-8-OH-DPAT by 50% at the given concentration. The lower portion of the table (P24 on)isthe ic2peptidestowardthe Cterminus. Thesepeptideswerelesseffective at decreasing specific high affinity binding of [ H]-8- OH-DPAT, and values given are percent of control at the given concentration. Values for intracellular cAMP are relative to FSK-stimulated control. All values for incorporation of γ-[ S]-GTP into Gi are percent of control. Nominal values for control binding were 400 fmoles/mg protein. Peptide Conc. (uM) % cont. ag, bound, (SEM) [cAMP] (SEM) GTP Incorp. (SEM) Control 100 (6) 100 (7) 5HT 21 (4) 168 (12) P11 7 50 (1) 87 (8) 100 (3) P21 15 52 (4) 122 (8) 158 (11) P22 16 51 (2) 71 (2) 128 (9) P23 30 50 (22) 42 (4) 188 (10) P24 10 94 (9) 45 (7) 146 (17) P25 30 87 (12) 64 (3) 126 (10) P26 30 75 (19) 100 (5) 111 (9) P27 30 95 (5) 132 (6) 130 (7) Peptide effect on forskolin (FSK) stimulated cAMP γ -[ S]-GTP incorporation for P12 and P13 production 261 ± 9 204 ± 15 185.5 ± 9 20 150 124 ± 6 112 ± 6 100 ± 4 FSK 5HT P21 P22 P23 P24 P25 P26 P27 Figure 3: ic2 peptide effect on forskolin-stimulated cAMP pro- duction, a measure of activated-G-protein regulation of adenylyl cyclase. Forskolin (FSK) stimulated cAMP production by adenylyl cyclase (AC) is in CHO cells expressing the human 5HT1aR. FSK (30 uM) is the control, which is set to 100%. All other treatments are Control 5HT (10- P12 (3∗ 5HT/P12 P13 (5∗ 5HT/P13 expressed as a percent of the control value. Peptide concentrations 6 M) 10-5 M) 10-3 M) are 30 uM. All treatments include isobutylmethylxanthine (IBMX) an inhibitor of the metabolism of cAMP by phosphodiesterase. Figure 4: P12 and P13-stimulated incorporation of γ-[ S]-GTP ErrorisexpressedasSEM. control is the basal amount of γ-[ S]-GTP incorporated into Gi in CHO cells expressing the human 5HT1aR set as 100%. The Y axis is the percent of specifically bound γ-[ S]-GTP. All other treatments are percents of the control value. Peptide concentrations are 30 uM. cAMP (Figure 3) may shed some light on this situation. Note ∗ C P< 0.01 P12 versus control; P< 0.01 5HT versus 5HT/P12. that GTP binding by Gi is an agonist-dependent process P13 versus control P< 0.01; 5HT versus 5HT/P13 P< 0.01. (see 5HT in the Figure); thus, when peptides increase GTP incorporation above control level, the implication is that the peptides are representing native loop regions under the on both sides are parallel for GTP incorporation (Figure 2) influence of agonist. and cAMP concentrations (Figure 3). Note that basal levels Figure 3 shows a parallel set of results whereby the of intracellular cAMP are quite low, and the experimental peptides’ ability to change intracellular cAMP concentrations protocol for these experiments involves artificially raising following coupling to Gi is determined (control is the FSK cAMP concentrations via stimulation of adenylyl cyclase stimulated level; agonist; e.g., 5HT activates Gi and lowers with forskolin (control) and comparison of peptide results cAMP levels below the control reading). Again, peptide P23, to that produced by the agonist serotonin. representing mid-loop residues, is most effective. In contrast to the results for GTP incorporation in Figure 2, the cAMP results have a smooth drop off in efficacy on both sides 3.2. Intracellular Loop 3 (ic3). H5HT1aR’s ic3 is much larger of P23. Overall, the trends peaking at P23 and declining than ic2 (about 130 amino acids); nevertheless, we did a very FSK Stimulated cAMP (%) Control (Sp.γ [ S]-GTP incorp) (%) International Journal of Peptides 5 Table 3: ic3 Peptide mimic coupling and signal transduction data. 124 ± 7 Summary of data generated for all ic3 experiments with P12 and 100 ± 5 100 ± 6 P13. P1 is included as a reference, from Hayataka et al., 1998 [49]. Nominal values for control agonist binding were 400 fmoles/mg 60 ± 4 75 protein. 41 ± 1 50 ∗ [35S]-γ-S-GTP % Inhibition of Agonist (%) Peptide incorporation% FSK-stimulated 24 ± 1 inhibition above conro cAMP P1 50 (3 uM) 30 (1 uM) 10 (10 uM) FSK 5HT P12 P13 5HT/P12 5HT/P13 P12 28 (30 uM) 24 (30 uM) 0 (30 uM) Treatment P13 50 (15 uM) 12 (30 uM) −24 (30 uM) Figure 5: P12 and P13 effect of forskolin-stimulated cAMP pro- duction forskolin (FSK) stimulated cAMP production by adenylyl cyclase (AC) in CHO cells expressing the human 5HT1aR. These current investigation we have presented further information experiments were a measure of second messenger regulation by about the H5HT1aR/Gi interface that should provide testable G protein. FSK is the control, which is set to 100%. All other treatments are expressed as a percent of the control value. Peptide hypotheses anticipating the ultimate structural analysis of concentrations are 30 uM. All treatments include isobutylmethyl the receptor. xanthine (IBMX), an inhibitor of the metabolism of cAMP. 5HT Data collected in previous and current experiments versus 5HT/P12 and 5HT versus 5HT/P13 P< 0.05. have implicated a role for ic2 in receptor coupling and G- protein activation. The N-terminus end of ic2, involving the sequence IALDRYWAITDPIDYV and including pep- limited number of comparisons at the N-terminal (TM5) tides P11 (previous work) and P21–P23 (current work), is region of ic3, continuing preliminary work [50–53]and important for coupling to the G protein. Evidence for this using the same approach as with ic2 by synthetically building includes presence of the highly conserved DRY sequence 10-MER’s two amino acids at a time from the parent (P1; for GPCR’s [51] and from the present study, IC50’s for the Table 1). Table 3 gives coupling and activation summaries peptides’ coupling capacity, with ranges from 7 to 30 uM. for the two peptides, P12 and P13 (Table 1). As with the Decay of G-protein coupling activity was observed as the ic2 peptides, the ic3 peptides, P12 and P13, give shallow, peptides progress towards the C-terminus of ic2 (P24– uM concentration-effect relationships (data not shown 27). As the amino acids seem to wane in importance for in graphical form as in Figure 1). For coupling, if 50% receptor coupling, they increase for part of the distance in is listed, then that is the IC 50; if another value is listed, importance for G-protein activation with its peak at the that is the maximum inhibition possible with the highest P23 amino acid stretch WAITDPIDYV. This is clearly shown soluble concentration. Both P12 and P13 produce small by the bell-shaped progression of the data bars for the but significant incorporation increases of GTP based upon incorporation of γ-[ S]-GTP into Gi (Figure 2). This can the amount of [ S]-GTP incorporated into Gi (Figure 4), be superimposed over the inverted bell-shaped depression while the outcomes for changes in intracellular cAMP for intracellular levels of FSK-stimulated cAMP production concentrations are more complex (Figure 5): for peptide P12, (Figure 3) following peptide treatment. intracellular cAMP concentration is not altered; unusually, The C-terminal end of ic2 consisting of the amino peptide P13 actually increases intracellular cAMP concen- acids RTPRPR may serve as an anchor, helping to hold the tration. A possible explanation for the combined results for amino terminal of ic2 in a favorable orientation for coupling P12 and P13 is given in the Discussion section. to the G protein [48]. Also interesting about the carboxy terminal end of ic2 is the presence of the 2 proline (P) residues separated by only 1 amino acid. These proline 4. Discussion residues in close proximity to each other introduce a kink H5HT1aR is linked to numerous important physiological in the receptor structure constraining its range of motion. and pathological processes. Additionally, the receptor is a These data demonstrates the clear role for H5HT1aR’s ic2 in close relative, not only of other 5HT1 type receptors, but coupling receptor to G protein, and toward the loop’s middle, also the beta adrenergic receptors and other GPCR’s [13, 42, G-protein activation. 55, 56]. Because of these characteristics, structural determi- For ic3, the inhibition of AC by Gi is an important nations of the receptor are crucial matters. Despite recent regulator of intracellular signal transduction. The current critical structural advances with the beta adrenergic receptors peptides tested, P12 and P13, differed in their ability to [10, 15, 16, 18], the 5HT1aR is uncrystallized and its struc- regulate this step in the cascade. P12 was unable to decrease ture awaits X-ray analysis [22]. the FSK-stimulated levels of cAMP (Figure 5). This is in In previous work [49, 50, 53, 54], we have demonstrated contrast to the action of 5HT which was able to significantly the utility of an agonist-based inhibition system associated decrease the FSK-stimulated levels of cAMP. P13 had the with signal transduction parameters to study interactions opposite effect; it increased cAMP concentrations (Figure 5)! of the receptor with its cognate G protein, Gi. In the This suggests that the two new amino acids (AD added to FSK stimulated cAMP (%) 6 International Journal of Peptides form P13) from ic3 are potentially at the beginning of a efficacious tertiary structure without the full loop being region of the loop which has negative regulatory properties present. It is crucial to point out that the parent ic3 peptide on Gi blunting its normal ability to regulate AC. It is (P1) contains the full TVKK sequence at its N-terminus. interesting to speculate about the differences in data from This sequence is part of the so-called Ric-8 [62, 63]region the γ-[ S]-GTP (Figure 4) incorporation assays and cAMP that has been shown in other GPCR as crucial to G-protein assays (Figure 5). P12 slightly increases GTP incorporation regulation. Significantly, the P1 relatives (P12 and P13) while P13 statistically does not. Thus, P12 activates Gi but under discussion in this communication are at a transition cAMP changes do not ensue. P13 does not activate Gi, point for this sequence; P12 contains the full TVKK stretch but a cAMP change occurs in the atypical direction. With while P13 has lost the T! One additional observation may the relatively small changes produced by these two peptides be pertinent. For GTP incorporation, for both peptides, the in both signaling measures, one possibility is experimental combination of peptide plus 5HT produces markedly greater error that has not been accounted for. It is possible that the incorporation than that produced by 5HT alone. This peptides are acting at some sites other than the proposed may suggest that 5HT and the peptides may be perturbing receptor-G-protein interface or that the process at the inter- separate sites on the receptor and/or G protein. face is more complex. In summary, this peptide mimic study for intracellular The most tantalizing possibility is that the newly explored loops 2 and 3 of the H5HT1aR was designed to examine region represented by P12 and P13 is the beginning of a which segments were involved in coupling and activation region of ic3 involved in coupling of receptor to G protein of Gi. The results reported here in combination with still capable of regulating Gi. Additionally, the perturbation previously reported work conclude that the amino terminal of Gi in this case involves different conformational changes ends of ic2 and ic3 are important for coupling the receptor that activate Gi but in a novel way. This would produce and G protein. The activation of G-protein peaks at P23 the opposite effect on cAMP concentration and would be (WAITDPIDYV) in ic2 (mid-loop). The activity is decreased equivalent to the downstream actions of an inverse agonist at as the structures move in either direction away from this the ligand binding site. Since 5HT1aR is capable of constitu- core sequence. The curious results of increased cAMP tive activity [25], inverse agonism is possible, and it will be concentrations caused by P13 suggests that the two new fascinating to see if the P12/P13 region is involved in this amino acids (AD) in P13 are the beginning of a new region activity once the crystal structure is available. In this context of ic3 which has negative regulatory properties on Gi. That then, P12 would represent a transitional region between is, the new region may be one that is not normally activated “normal” and “atypical” Gi regulation while P13 is in the by agonists; however, in the presence of inverse agonists and atypical subregion. the different conformational changes they produce, the new While the data support this region’s (P12/P13) role in region may couple to and activate Gi in a way that regulates receptor-G-protein coupling, the peptides’ ability to uncou- AC in a way we define as inverse agonism. The combined ple declines relative to previously studied peptides whose results with H5HT1aR ic2 and ic3 peptides should lead structures represent segments closer to ic3’s N-terminus. to testable crystallographic hypotheses with drugs having P12 and 13 are beyond (toward the C-terminus) the key differential intrinsic activities. Beyond the final judgment of RFRI region of P1 previously identified as key to that these peptide probes in the structural sense, the information part of 5HT1aR’s ic3-N-terminus responsible for G-protein produced may be useful as independent pharmacological activation [50]. observations. Pragmatic implications of the work may be Varrault et al. [48] demonstrated that the C-terminal relevant in a framework where the multiple, differential section of i3 is involved in G protein coupling and regulation. activities of the peptides can be used by medicinal chemists So, if our work can be interpreted to mean that peak coupling to build unique pharmacological agents targeting unutilized and activating properties are associated with ic3’s N-terminal sites at the receptor-G-protein interface. residues and Varrault’s work can be interpreted to mean that peak coupling and activating properties are associated with ic3’s C-terminal residues, then what role will hold for the vast Acknowledgments internal region of the loop in 5HT1aR? GPCR ic3’s are vari- able in size in rhodopsin versus 5HT1aR and BAR’s, which The authors would like to express deepest appreciation to the have larger ic3 loops (at least twice the size of rhodopsin’s Department of Biomedical and Pharmaceutical Sciences, the ic3). It would be meaningful to extend this peptide approach Skaggs School of Pharmacy, the College of Health Professions into the midloop region of H5HT1a’s ic3, and then as a crys- and Biomedical Sciences, all of The University of Montana tal structure becomes available the comparisons of 5HT1aR (UM), Missoula. Without the resources and human support loop function with BAR and rhodopsin will be fascinating. of these units and The University, the project could not Neither of the peptides (P12 and P13) are as potent have occurred. Special thanks to Dr. David Freeman for as 5HT at incorporating GTP into Gi . It is possible that his editorial assistance. Research was conducted under the multiple regions are responsible for G-protein activation, generous and essential sponsorship of the following NIH and the individual peptides mimic only part of this structure Grants: UM-CHPBS Endowment Fund Program NIH S21- [61], thereby producing a diminished effect relative to 5HT. MD000236, RR10169 and GM/OD 54302-01/02, and P20 Also, a given peptide region, even one that is absolutely RR 15583 to the UM COBRE Center for Structural and critical in the native structure, may not have the most Functional Neurosciences from NCRR. International Journal of Peptides 7 References [19] V. P. Jaakola, M. T. Griffith,M.A.Hansonetal., “The 2.6 angstrom crystal structure of a human A2A adenosine recep- [1] E. C. Azmitia, “Serotonin and brain: evolution, neuroplastic- tor bound to an antagonist,” Science, vol. 322, no. 5905, pp. ity, and homeostasis,” International Review of Neurobiology, 1211–1217, 2008. vol. 77, pp. 31–56, 2006. [20] B. Wu, E. Y. T. Chien, C. D. Mol et al., “Structures of the [2] M.Filip andM.Bader,“Overview on 5-HT receptorsand CXCR4 chemokine GPCR with small-molecule and cyclic their role in physiology and pathology of the central nervous peptide antagonists,” Science, vol. 330, no. 6007, pp. 1066– system,” Pharmacological Reports, vol. 61, no. 5, pp. 761–777, 1071, 2010. 2009. [21] E. Y. T. Chien, W. Liu, Q. Zhao et al., “Structure of the human dopamine D3 receptor in complex with a D2/D3 selective [3] D. E. Nichols and C. D. Nichols, “Serotonin receptors,” Chem- antagonist,” Science, vol. 330, no. 6007, pp. 1091–1095, 2010. ical Reviews, vol. 108, no. 5, pp. 1614–1641, 2008. [22] S. Topiol and M. Sabio, “X-ray structure breakthroughs in the [4] N. M. Barnes and T. Sharp, “A review of central 5-HT receptors GPCR transmembrane region,” Biochemical Pharmacology, and their function,” Neuropharmacology,vol. 38, no.8,pp. vol. 78, no. 1, pp. 11–20, 2009. 1083–1152, 1999. [23] J. R. Raymond, Y. V. Mukhin, T. W. Gettys, and M. N. Gar- [5] D. Hoyer, D. E. Clarke, J. R. Fozard et al., “International novskaya, “The recombinant 5-HT(1A) receptor: G protein Union of Pharmacology classification of receptors for 5-hy- coupling and signalling pathways,” British Journal of Pharma- droxytryptamine (serotonin),” Pharmacological Reviews, vol. cology, vol. 127, no. 8, pp. 1751–1764, 1999. 46, no. 2, pp. 157–203, 1994. [24] J. R. Raymond, Y. V. Mukhin, A. Gelasco et al., “Multiplicity [6] P. J. Goadsby, A. R. Charbit, A. P. Andreou, S. Akerman, and of mechanisms of serotonin receptor signal transduction,” P. R. Holland, “Neurobiology of migraine,” Neuroscience, vol. Pharmacology and Therapeutics, vol. 92, no. 2-3, pp. 179–212, 161, no. 2, pp. 327–341, 2009. [7] K. K. Parker, “Involvement of adipokines in migraine head- [25] J. C. Martel,A.M.Ormiere,N.Leduc,M.B.Assie, D. Cussac, ache,” in Extracellular & Intracellular Signaling,J.D.Adams and A. Newman-Tancredi, “Native rat hippocampal 5-HT 1A and K. K. Parker, Eds., Royal Society of Chemistry, Cambridge, receptors show constitutive activity,” Molecular Pharmacology, UK, 2011. vol. 71, no. 3, pp. 638–643, 2007. [26] J. Guptarak, A. Selvamani, and L. Uphouse, “GABAA-5-HT [8] H. R. Bourne, “G-proteins and GPCRs: from the beginning,” 1A receptor interaction in the mediobasal hypothalamus,” Brain Ernst Schering Foundation symposium proceedings,no. 2, pp. Research, vol. 1027, no. 1-2, pp. 144–150, 2004. 1–21, 2006. [27] E. B. Russo, A. Burnett, B. Hall, and K. K. Parker, “Agonistic [9] S. C. Sinha and S. R. Sprang, “Structures, mechanism, regula- properties of cannabidiol at 5-HT receptors,” Neurochemical 1A tion and evolution of class III nucleotidyl cyclases,” Reviews of Research, vol. 30, no. 8, pp. 1037–1043, 2005. Physiology, Biochemistry and Pharmacology, vol. 157, pp. 105– [28] C. P. Muller,R.J.Carey,J.P.Huston, andM.A.deSouza Silva, 140, 2006. “Serotonin and psychostimulant addiction: focus on 5-HT - 1A [10] B. K. Kobilka, “G protein coupled receptor structure and receptors,” Progress in Neurobiology, vol. 81, no. 3, pp. 133– activation,” Biochimica et Biophysica Acta, vol. 1768, no. 4, pp. 178, 2007. 794–807, 2007. [29] C. Jonnakuty and C. Gragnoli, “What do we know about [11] B. K. Kobilka and X. Deupi, “Conformational complexity of serotonin?” Journal of Cellular Physiology, vol. 217, no. 2, pp. G-protein-coupled receptors,” Trends in Pharmacological Sci- 301–306, 2008. ences, vol. 28, no. 8, pp. 397–406, 2007. [30] D. Kozaric-Kovacic, “Psychopharmacotherapy of posttrau- [12] R. J. Lefkowitz, J. P. Sun, and A. K. Shukla, “A crystal clear matic stress disorder,” Croatian Medical Journal, vol. 49, no. view of the β -adrenergic receptor,” Nature Biotechnology, vol. 4, pp. 459–475, 2008. 26, no. 2, pp. 189–191, 2008. [31] J. L. Rausch, M. E. Johnson, K. E. Kasik, and S. M. Stahl, “Tem- perature regulation in depression: functional 5HT1A receptor [13] D. M. Rosenbaum, S. G. F. Rasmussen, and B. K. Kobilka, adaptation differentiates antidepressant response,” Neuropsy- “The structure and function of G-protein-coupled receptors,” chopharmacology, vol. 31, no. 10, pp. 2274–2280, 2006. Nature, vol. 459, no. 7245, pp. 356–363, 2009. [32] E. Akimova, R. Lanzenberger, and S. Kasper, “The serotonin- [14] J. M. Baldwin, “Structure and function of receptors coupled to 1A receptor in anxiety disorders,” Biological Psychiatry, vol. 66, G proteins,” Current Opinion in Cell Biology,vol. 6, no.2,pp. no. 7, pp. 627–635, 2009. 180–190, 1994. [33] G. S. Kranz, S. Kasper, and R. Lanzenberger, “Reward and the [15] V. Cherezov,D.M.Rosenbaum,M.A.Hansonetal., “High- serotonergic system,” Neuroscience, vol. 166, no. 4, pp. 1023– -adren- resolution crystal structure of an engineered human β 1035, 2010. ergic G protein-coupled receptor,” Science, vol. 318, no. 5854, [34] L. A. Catapano and H. K. Manji, “G protein-coupled receptors pp. 1258–1265, 2007. in major psychiatric disorders,” Biochimica et Biophysica Acta, [16] S. G. F. Rasmussen, H. J. Choi, D. M. Rosenbaum et al., “Crys- vol. 1768, no. 4, pp. 976–993, 2007. tal structure of the human β adrenergic G-protein-coupled [35] B. le Francois, M. Czesak, D. Steubl, and P. R. Albert, “Tran- receptor,” Nature, vol. 450, no. 7168, pp. 383–387, 2007. scriptional regulation at a HTR1A polymorphism associated [17] D. M. Rosenbaum, V. Cherezov, M. A. Hanson et al., “GPCR with mental illness,” Neuropharmacology,vol. 55, no.6,pp. engineering yields high-resolution structural insights into β - 2 977–985, 2008. adrenergic receptor function,” Science, vol. 318, no. 5854, pp. [36] L. B. Resstel,R.F.Tavares,S.F.Lisboa, S. R. Joca,F.M.Correa, 1266–1273, 2007. and F. S. Guimaraes, “5-HT receptors are involved in the 1A [18] D. M. Rosenbaum, C. Zhang, J. A. Lyons et al., “Structure and cannabidiol-induced attenuation of behavioral & cardiovascu- lar responses to acute restraint stress in rats,” British Journal of function of an irreversible agonist-β adrenoceptor complex,” Nature, vol. 469, no. 7329, pp. 236–240, 2011. Pharmacology, vol. 156, no. 1, pp. 181–188, 2009. 8 International Journal of Peptides [37] J. Savitz, I. Lucki, and W. C. Drevets, “5-HT receptor func- [53] H. V. Thiagaraj, T. C. Ortiz, M. C. Devereaux Jr., B. Seaver, B. 1A tion in major depressive disorder,” Progress in Neurobiology, Hall, and K. K. Parker, “Regulation of G proteins by human vol. 88, no. 1, pp. 17–31, 2009. 5-HT receptor TM3/i2 and TM5/i3 loop peptides,” Neuro- 1A chemistry International, vol. 50, no. 1, pp. 109–118, 2007. [38] G. V. Carr and I. Lucki, “The role of serotonin receptor sub- types in treating depression: a review of animal studies,” Psy- [54] B. Hall, A. Burnett, A. Christians et al., “Thermodynamics of chopharmacology, vol. 213, no. 2-3, pp. 265–287, 2011. peptide and non-peptide interactions with the human 5HT 1A receptor,” Pharmacology, vol. 86, no. 1, pp. 6–14, 2010. [39] P. N. Yadav, A. I. Abbas, M. S. Farrell et al., “The presynaptic component of the serotonergic system is required for cloza- [55] A. Fargin, J. R. Raymond, M. J. Lohse, B. K. Kobilka, M. G. pine’s efficacy,” Neuropsychopharmacology,vol. 36, no.3,pp. Caron, and R. J. Lefkowitz, “The genomic clone G-21 which 638–651, 2011. resembles a β-adrenergic receptor sequence encodes the 5- HT receptor,” Nature, vol. 335, no. 6188, pp. 358–360, 1988. [40] J. P. Changeux and S. J. Edelstein, “Allosteric mechanisms of 1A signal transduction,” Science, vol. 308, no. 5727, pp. 1424– [56] B. K. Kobilka, T. Frielle, S. Collins et al., “An intronless gene 1428, 2005. encoding a potential member of the family of receptors cou- pled to guanine nucleotide regulatory proteins,” Nature, vol. [41] J. H. Turner, M. N. Garnovskaya, and J. R. Raymond, “Sero- 329, no. 6134, pp. 75–79, 1987. tonin 5-HT receptor stimulates c-Jun N-terminal kinase 1A and induces apoptosis in Chinese hamster ovary fibroblasts,” [57] M. M. Bradford, “A rapid and sensitive method for the quan- Biochimica et Biophysica Acta, vol. 1773, no. 3, pp. 391–399, titation of microgram quantities of protein utilizing the prin- 2007. ciple of protein dye binding,” Analytical Biochemistry, vol. 72, no. 1-2, pp. 248–254, 1976. [42] M. C. Lagerstrom and H. B. Schioth, “Structural diversity of G protein-coupled receptors and significance for drug discov- [58] T. Wieland and K. H. Jakobs, “[1] Measurement of receptor- ery,” Nature reviews. Drug Discovery, vol. 7, no. 4, pp. 339–357, stimulated guanosine 5 -O-(γ-thio)triphosphate binding by G 2008. proteins,” Methods in Enzymology, vol. 237, pp. 3–13, 1994. [43] M. J. Millan, P. Marin, J. Bockaert, and C. Mannoury la Cour, [59] M. E. Maguire, P. M. van Arsdale, and A. G. Gilman, “An “Signaling at G-protein-coupled serotonin receptors: recent agonist specific effect of guanine nucleotides on binding to the advances and future research directions,” Trends in Pharma- β adrenergic receptor,” Molecular Pharmacology, vol. 12, no. 2, cological Sciences, vol. 29, no. 9, pp. 454–464, 2008. pp. 335–339, 1976. [44] B. Sjgren,L.L.Blazer, andR.R.Neubig, “Regulatorsof [60] S. J. Peroutka, R. M. Lebovitz, and S. H. Snyder, “Sero- G protein signaling proteins as targets for drug discovery,” tonin receptor binding sites affected differentially by guanine Progress in Molecular Biology and Translational Science, vol. 91, nucleotides,” Molecular Pharmacology, vol. 16, no. 3, pp. 700– no. C, pp. 81–119, 2010. 708, 1979. [45] J. A. Allen and B. L. Roth, “Strategies to discover unexpected [61] S. Kalipatnapu and A. Chattopadhyay, “Membrane organiza- targets for drugs active at G protein-coupled receptors,” An- tion and function of the serotonin receptor,” Cellular and 1A nual Review of Pharmacology and Toxicology, vol. 51, pp. 117– Molecular Neurobiology, vol. 27, no. 8, pp. 1097–1116, 2007. 144, 2011. [62] G. G. Tall, A. M. Krumins, and A. G. Gilman, “Mammalian [46] S. Ganguly, A. H. A. Clayton, and A. Chattopadhyay, “Orga- Ric-8A (synembryn) is a heterotrimeric Gα protein guanine nization of higher-order oligomers of the serotonin receptor nucleotide exchange factor,” The Journal of Biological Chem- 1A explored utilizing homo-FRET in live cells,” Biophysical Jour- istry, vol. 278, no. 10, pp. 8356–8362, 2003. nal, vol. 100, no. 2, pp. 361–368, 2011. [63] C. J. Thomas, G. G. Tall, A. Adhikari, and S. R. Sprang, “Ric- [47] A. Ivetac and J. Andrew McCammon, “Mapping the druggable 8A catalyzes guanine nucleotide exchange on Gαi1 bound to allosteric space of g-protein coupled receptors: a fragment- the GPR/GoLoco exchange inhibitor AGS3,” The Journal of based molecular dynamics approach,” Chemical Biology and Biological Chemistry, vol. 283, no. 34, pp. 23150–23160, 2008. Drug Design, vol. 76, no. 3, pp. 201–217, 2010. [48] A. Varrault, Dung Le Nguyen, S. McClue, B. Harris, P. Jouin, and J. Bockaert, “5-Hydroxytryptamine receptor synthetic 1A peptides: mechanisms of adenylyl cyclase inhibition,” The Journal of Biological Chemistry, vol. 269, no. 24, pp. 16720– 16725, 1994. [49] K. Hayataka, M. F. O’Connor, N. Kinzler, J. T. Weber, and K. K. Parker, “A bioactive peptide from the transmembrane 5-intracellular loop 3 region of the human 5HT receptor,” 1A Biochemistry and Cell Biology, vol. 76, no. 4, pp. 657–660, 1998. [50] T. C. Ortiz, M. C. Devereaux, and K. K. Parker, “Structural variants of a human 5-HT receptor intracellular loop 3 1A peptide,” Pharmacology, vol. 60, no. 4, pp. 195–202, 2000. [51] N. Kushwaha, S. C. Harwood, A. M. Wilson et al., “Molecular determinants in the second intracellular loop of the 5-hy- droxytryptamine-1A receptor for G-protein coupling,” Molec- ular Pharmacology, vol. 69, no. 5, pp. 1518–1526, 2006. [52] A. O. Shpakov and M. N. Pertseva, “Molecular mechanisms for the effect of mastoparan on G proteins in tissues of verte- brates and invertebrates,” Bulletin of Experimental Biology and Medicine, vol. 141, no. 3, pp. 302–306, 2006. 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