title: The hydrophobic amino acids in putative helix 8 in carboxy-terminus of histamine H(3) receptor are involved in receptor-G-protein coupling.
abstract: Functional roles of putative helix 8 in the carboxy-terminal tail of the human histamine H(3) 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, [((35))S] 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 H(3) 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 H(3) receptor activity, presumably by stabilizing the helix to the plasma membrane.
authors.affiliation: Department of Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Miyagi 980-8575, Japan. atsuok@yamaguchi-u.ac.jp
authors.forename: Atsuo
authors.initials: A
authors.lastname: Kuramasu
authors.forename: Jun
authors.initials: J
authors.lastname: Sukegawa
authors.forename: Takeya
authors.initials: T
authors.lastname: Sato
authors.forename: Eiko
authors.initials: E
authors.lastname: Sakurai
authors.forename: Takehiko
authors.initials: T
authors.lastname: Watanabe
authors.forename: Teruyuki
authors.initials: T
authors.lastname: Yanagisawa
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Yanai
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology; D005434:Flow Cytometry; D019204:GTP-Binding Proteins / Q000378:metabolism*; D057809:HEK293 Cells; D006632:Histamine / Q000378:metabolism; D017442:Histamine Agonists / Q000494:pharmacology*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010880:Piperidines / Q000494:pharmacology; D010957:Plasmids; D011485:Protein Binding; D017433:Protein Structure, Secondary; D018100:Receptors, Histamine H3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics*; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: D000596:Amino Acids; D017442:Histamine Agonists; D010880:Piperidines; D018100:Receptors, Histamine H3; D006632:Histamine; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; C052075:thioperamide