The hydrophobic amino acids in putative helix 8 in carboxy-terminus of histamine H(3) receptor are involved in receptor-G-protein coupling |
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Authors: | Kuramasu Atsuo Sukegawa Jun Sato Takeya Sakurai Eiko Watanabe Takehiko Yanagisawa Teruyuki Yanai Kazuhiko |
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Affiliation: | a Department of Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Miyagi 980-8575, Japanb Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Miyagi 980-8575, Japan |
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Abstract: | Functional roles of putative helix 8 in the carboxy-terminal tail of the human histamine H3 receptor were investigated using deleted and alanine-substituted mutant receptors. While the deletion of the carboxy-terminal tail did not decrease the total expression level, surface expression, or ligand binding affinity, the agonist-stimulated cAMP response, [35S] GTPγS binding, and MAPK activation were totally abolished. The receptor lacking the carboxy-terminal tail also failed to respond to an inverse agonist, thioperamide, suggesting that the carboxy-terminal tail is involved in the regulation of receptor activity by changing G-protein coupling with the receptor. Site-directed mutagenesis revealed that hydrophobic amino acids in the putative helix 8 such as phenylalanines at position 419 (F7.60) and 423 (F7.64) or leucines at 426 (L7.67) and 427 (L7.68) were important for the agonist-induced activation of H3 receptor. Substitution of F7.60 also resulted in a receptor that was less responsive to inactivation by the inverse agonist, implying the existence of an intermediate conformation that can be either activated or inactivated. Our results suggest that hydrophobic interface of putative helix 8 is important for the regulation of H3 receptor activity, presumably by stabilizing the helix to the plasma membrane. |
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Keywords: | GPCR, G-protein coupled receptor H3R, histamine H3 receptor HEK, human embryonic kidney CLIC4, chloride intracellular channel 4 DMEM, Dulbecco's modified Eagle's medium PBS, phosphate buffered saline PAGE, polyacrylamide gel electrophoresis RAMH, R-(&minus )-α-methylhistamine CRE, cAMP response element BSA, bovine serum albumin DRiP78, dopamine receptor-interacting protein 78 TMD, transmembrane domain WT, wild-type ERK, extracellular signal-regulated kinase |
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