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 Regulation of serotonergic (5-hydroxytryptamine; 5HT) function in animals impacts numerous physiological and pathological processes [1]. 5HT is broadly represented in biological systems as a regulator and modulator via nervous, hormonal, and autacoidal means [2–5]. For example, serotonin [6] has been implicated in the pathophysiology of migraine. This association with migraine is shared with many other factors including adipokines such as leptin; hypothalamic hormones, Orexin A and B (also known appetite regulators as is 5HT); numerous neurotransmitters [7]; autacoids; hormones, and ions like calcium, and magnesium. The range of biological molecules that interact with serotonergic processes suggests that various signaling pathways may be shared, and that the potential for dynamic, collaborative regulation exists. Better understanding of the molecular basis underlying these signaling processes is not only critical to greater fundamental knowledge but to therapeutic development. Various
References
[1]
E. C. Azmitia, “Serotonin and brain: evolution, neuroplasticity, and homeostasis,” International Review of Neurobiology, vol. 77, pp. 31–56, 2006.
[2]
M. Filip and M. Bader, “Overview on 5-HT receptors and their role in physiology and pathology of the central nervous system,” Pharmacological Reports, vol. 61, no. 5, pp. 761–777, 2009.
[3]
D. E. Nichols and C. D. Nichols, “Serotonin receptors,” Chemical Reviews, vol. 108, no. 5, pp. 1614–1641, 2008.
[4]
N. M. Barnes and T. Sharp, “A review of central 5-HT receptors and their function,” Neuropharmacology, vol. 38, no. 8, pp. 1083–1152, 1999.
[5]
D. Hoyer, D. E. Clarke, J. R. Fozard et al., “International Union of Pharmacology classification of receptors for 5-hydroxytryptamine (serotonin),” Pharmacological Reviews, vol. 46, no. 2, pp. 157–203, 1994.
[6]
P. J. Goadsby, A. R. Charbit, A. P. Andreou, S. Akerman, and P. R. Holland, “Neurobiology of migraine,” Neuroscience, vol. 161, no. 2, pp. 327–341, 2009.
[7]
K. K. Parker, “Involvement of adipokines in migraine headache,” in Extracellular & Intracellular Signaling, J. D. Adams and K. K. Parker, Eds., Royal Society of Chemistry, Cambridge, UK, 2011.
[8]
H. R. Bourne, “G-proteins and GPCRs: from the beginning,” Ernst Schering Foundation symposium proceedings, no. 2, pp. 1–21, 2006.
[9]
S. C. Sinha and S. R. Sprang, “Structures, mechanism, regulation and evolution of class III nucleotidyl cyclases,” Reviews of Physiology, Biochemistry and Pharmacology, vol. 157, pp. 105–140, 2006.
[10]
B. K. Kobilka, “G protein coupled receptor structure and activation,” Biochimica et Biophysica Acta, vol. 1768, no. 4, pp. 794–807, 2007.
[11]
B. K. Kobilka and X. Deupi, “Conformational complexity of G-protein-coupled receptors,” Trends in Pharmacological Sciences, vol. 28, no. 8, pp. 397–406, 2007.
[12]
R. J. Lefkowitz, J. P. Sun, and A. K. Shukla, “A crystal clear view of the β2-adrenergic receptor,” Nature Biotechnology, vol. 26, no. 2, pp. 189–191, 2008.
[13]
D. M. Rosenbaum, S. G. F. Rasmussen, and B. K. Kobilka, “The structure and function of G-protein-coupled receptors,” Nature, vol. 459, no. 7245, pp. 356–363, 2009.
[14]
J. M. Baldwin, “Structure and function of receptors coupled to G proteins,” Current Opinion in Cell Biology, vol. 6, no. 2, pp. 180–190, 1994.
[15]
V. Cherezov, D. M. Rosenbaum, M. A. Hanson et al., “High-resolution crystal structure of an engineered human β2-adrenergic G protein-coupled receptor,” Science, vol. 318, no. 5854, pp. 1258–1265, 2007.
[16]
S. G. F. Rasmussen, H. J. Choi, D. M. Rosenbaum et al., “Crystal structure of the human β2 adrenergic G-protein-coupled receptor,” Nature, vol. 450, no. 7168, pp. 383–387, 2007.
[17]
D. M. Rosenbaum, V. Cherezov, M. A. Hanson et al., “GPCR engineering yields high-resolution structural insights into β2-adrenergic receptor function,” Science, vol. 318, no. 5854, pp. 1266–1273, 2007.
[18]
D. M. Rosenbaum, C. Zhang, J. A. Lyons et al., “Structure and function of an irreversible agonist-β2 adrenoceptor complex,” Nature, vol. 469, no. 7329, pp. 236–240, 2011.
[19]
V. P. Jaakola, M. T. Griffith, M. A. Hanson et al., “The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist,” Science, vol. 322, no. 5905, pp. 1211–1217, 2008.
[20]
B. Wu, E. Y. T. Chien, C. D. Mol et al., “Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists,” Science, vol. 330, no. 6007, pp. 1066–1071, 2010.
[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 antagonist,” Science, vol. 330, no. 6007, pp. 1091–1095, 2010.
[22]
S. Topiol and M. Sabio, “X-ray structure breakthroughs in the GPCR transmembrane region,” Biochemical Pharmacology, vol. 78, no. 1, pp. 11–20, 2009.
[23]
J. R. Raymond, Y. V. Mukhin, T. W. Gettys, and M. N. Garnovskaya, “The recombinant 5-HT(1A) receptor: G protein coupling and signalling pathways,” British Journal of Pharmacology, vol. 127, no. 8, pp. 1751–1764, 1999.
[24]
J. R. Raymond, Y. V. Mukhin, A. Gelasco, et al., “Multiplicity of mechanisms of serotonin receptor signal transduction,” Pharmacology and Therapeutics, vol. 92, no. 2-3, pp. 179–212, 2001.
[25]
J. C. Martel, A. M. Ormiere, N. Leduc, M. B. Assie, D. Cussac, and A. Newman-Tancredi, “Native rat hippocampal 5-H receptors show constitutive activity,” Molecular Pharmacology, vol. 71, no. 3, pp. 638–643, 2007.
[26]
J. Guptarak, A. Selvamani, and L. Uphouse, “GABAA-5-H receptor interaction in the mediobasal hypothalamus,” Brain Research, vol. 1027, no. 1-2, pp. 144–150, 2004.
[27]
E. B. Russo, A. Burnett, B. Hall, and K. K. Parker, “Agonistic properties of cannabidiol at 5-H receptors,” Neurochemical Research, vol. 30, no. 8, pp. 1037–1043, 2005.
[28]
C. P. Muller, R. J. Carey, J. P. Huston, and M. A. de Souza Silva, “Serotonin and psychostimulant addiction: focus on 5-H -receptors,” Progress in Neurobiology, vol. 81, no. 3, pp. 133–178, 2007.
[29]
C. Jonnakuty and C. Gragnoli, “What do we know about serotonin?” Journal of Cellular Physiology, vol. 217, no. 2, pp. 301–306, 2008.
[30]
D. Kozaric-Kovacic, “Psychopharmacotherapy of posttraumatic stress disorder,” Croatian Medical Journal, vol. 49, no. 4, pp. 459–475, 2008.
[31]
J. L. Rausch, M. E. Johnson, K. E. Kasik, and S. M. Stahl, “Temperature regulation in depression: functional 5HT1A receptor adaptation differentiates antidepressant response,” Neuropsychopharmacology, vol. 31, no. 10, pp. 2274–2280, 2006.
[32]
E. Akimova, R. Lanzenberger, and S. Kasper, “The serotonin-1A receptor in anxiety disorders,” Biological Psychiatry, vol. 66, no. 7, pp. 627–635, 2009.
[33]
G. S. Kranz, S. Kasper, and R. Lanzenberger, “Reward and the serotonergic system,” Neuroscience, vol. 166, no. 4, pp. 1023–1035, 2010.
[34]
L. A. Catapano and H. K. Manji, “G protein-coupled receptors in major psychiatric disorders,” Biochimica et Biophysica Acta, vol. 1768, no. 4, pp. 976–993, 2007.
[35]
B. le Francois, M. Czesak, D. Steubl, and P. R. Albert, “Transcriptional regulation at a HTR1A polymorphism associated with mental illness,” Neuropharmacology, vol. 55, no. 6, pp. 977–985, 2008.
[36]
L. B. Resstel, R. F. Tavares, S. F. Lisboa, S. R. Joca, F. M. Correa, and F. S. Guimaraes, “5-H receptors are involved in the cannabidiol-induced attenuation of behavioral & cardiovascular responses to acute restraint stress in rats,” British Journal of Pharmacology, vol. 156, no. 1, pp. 181–188, 2009.
[37]
J. Savitz, I. Lucki, and W. C. Drevets, “5-H receptor function in major depressive disorder,” Progress in Neurobiology, vol. 88, no. 1, pp. 17–31, 2009.
[38]
G. V. Carr and I. Lucki, “The role of serotonin receptor subtypes in treating depression: a review of animal studies,” Psychopharmacology, vol. 213, no. 2-3, pp. 265–287, 2011.
[39]
P. N. Yadav, A. I. Abbas, M. S. Farrell et al., “The presynaptic component of the serotonergic system is required for clozapine's efficacy,” Neuropsychopharmacology, vol. 36, no. 3, pp. 638–651, 2011.
[40]
J. P. Changeux and S. J. Edelstein, “Allosteric mechanisms of signal transduction,” Science, vol. 308, no. 5727, pp. 1424–1428, 2005.
[41]
J. H. Turner, M. N. Garnovskaya, and J. R. Raymond, “Serotonin 5-H receptor stimulates c-Jun N-terminal kinase and induces apoptosis in Chinese hamster ovary fibroblasts,” Biochimica et Biophysica Acta, vol. 1773, no. 3, pp. 391–399, 2007.
[42]
M. C. Lagerstrom and H. B. Schioth, “Structural diversity of G protein-coupled receptors and significance for drug discovery,” Nature reviews. Drug Discovery, vol. 7, no. 4, pp. 339–357, 2008.
[43]
M. J. Millan, P. Marin, J. Bockaert, and C. Mannoury la Cour, “Signaling at G-protein-coupled serotonin receptors: recent advances and future research directions,” Trends in Pharmacological Sciences, vol. 29, no. 9, pp. 454–464, 2008.
[44]
B. Sjgren, L. L. Blazer, and R. R. Neubig, “Regulators of G protein signaling proteins as targets for drug discovery,” Progress in Molecular Biology and Translational Science, vol. 91, no. C, pp. 81–119, 2010.
[45]
J. A. Allen and B. L. Roth, “Strategies to discover unexpected targets for drugs active at G protein-coupled receptors,” Annual Review of Pharmacology and Toxicology, vol. 51, pp. 117–144, 2011.
[46]
S. Ganguly, A. H. A. Clayton, and A. Chattopadhyay, “Organization of higher-order oligomers of the receptor explored utilizing homo-FRET in live cells,” Biophysical Journal, vol. 100, no. 2, pp. 361–368, 2011.
[47]
A. Ivetac and J. Andrew McCammon, “Mapping the druggable allosteric space of g-protein coupled receptors: a fragment-based molecular dynamics approach,” Chemical Biology and 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- receptor synthetic 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 5H receptor,” 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-H receptor intracellular loop 3 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-hydroxytryptamine-1A receptor for G-protein coupling,” Molecular 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 vertebrates and invertebrates,” Bulletin of Experimental Biology and Medicine, vol. 141, no. 3, pp. 302–306, 2006.
[53]
H. V. Thiagaraj, T. C. Ortiz, M. C. Devereaux Jr., B. Seaver, B. Hall, and K. K. Parker, “Regulation of G proteins by human 5-H receptor TM3/i2 and TM5/i3 loop peptides,” Neurochemistry International, vol. 50, no. 1, pp. 109–118, 2007.
[54]
B. Hall, A. Burnett, A. Christians et al., “Thermodynamics of peptide and non-peptide interactions with the human 5H receptor,” Pharmacology, vol. 86, no. 1, pp. 6–14, 2010.
[55]
A. Fargin, J. R. Raymond, M. J. Lohse, B. K. Kobilka, M. G. Caron, and R. J. Lefkowitz, “The genomic clone G-21 which resembles a β-adrenergic receptor sequence encodes the 5-H receptor,” Nature, vol. 335, no. 6188, pp. 358–360, 1988.
[56]
B. K. Kobilka, T. Frielle, S. Collins, et al., “An intronless gene encoding a potential member of the family of receptors coupled to guanine nucleotide regulatory proteins,” Nature, vol. 329, no. 6134, pp. 75–79, 1987.
[57]
M. M. Bradford, “A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding,” Analytical Biochemistry, vol. 72, no. 1-2, pp. 248–254, 1976.
[58]
T. Wieland and K. H. Jakobs, “[1] Measurement of receptor-stimulated guanosine 5′-O-(γ-thio)triphosphate binding by G proteins,” Methods in Enzymology, vol. 237, pp. 3–13, 1994.
[59]
M. E. Maguire, P. M. van Arsdale, and A. G. Gilman, “An agonist specific effect of guanine nucleotides on binding to the β adrenergic receptor,” Molecular Pharmacology, vol. 12, no. 2, pp. 335–339, 1976.
[60]
S. J. Peroutka, R. M. Lebovitz, and S. H. Snyder, “Serotonin receptor binding sites affected differentially by guanine nucleotides,” Molecular Pharmacology, vol. 16, no. 3, pp. 700–708, 1979.
[61]
S. Kalipatnapu and A. Chattopadhyay, “Membrane organization and function of the receptor,” Cellular and Molecular Neurobiology, vol. 27, no. 8, pp. 1097–1116, 2007.
[62]
G. G. Tall, A. M. Krumins, and A. G. Gilman, “Mammalian Ric-8A (synembryn) is a heterotrimeric Gα protein guanine nucleotide exchange factor,” The Journal of Biological Chemistry, vol. 278, no. 10, pp. 8356–8362, 2003.
[63]
C. J. Thomas, G. G. Tall, A. Adhikari, and S. R. Sprang, “Ric-8A catalyzes guanine nucleotide exchange on Gαi1 bound to the GPR/GoLoco exchange inhibitor AGS3,” The Journal of Biological Chemistry, vol. 283, no. 34, pp. 23150–23160, 2008.
