title: The effect of mutations in the DRY motif on the constitutive activity and structural instability of the histamine H(2) receptor.
abstract: In previous studies we showed that the wild-type histamine H(2) receptor stably expressed in Chinese hamster ovary cells is constitutively active. Because constitutive activity of the H(2) receptor is already found at low expression levels (300 fmol/mg protein) this receptor is a relatively unique member of the G-protein-coupled receptor (GPCR) family and a useful tool for studying GPCR activation. In this study the role of the highly conserved DRY motif in activation of the H(2) receptor was investigated. Mutation of the aspartate 115 residue in this motif resulted in H(2) receptors with high constitutive activity, increased agonist affinity, and increased signaling properties. In addition, the mutant receptors were shown to be highly structurally instable. Mutation of the arginine 116 residue in the DRY motif resulted also in a highly structurally instable receptor; expression of the receptor could only be detected after stabilization with either an agonist or inverse agonist. Moreover, the agonist affinity at the Arg-116 mutant receptors was increased, whereas the signal transduction properties of these receptors were decreased. We conclude that the Arg-116 mutant receptors can adopt an active conformation but have a decreased ability to couple to or activate the G(s)-protein. This study examines the pivotal role of the aspartate and arginine residues of the DRY motif in GPCR function. Disruption of receptor stabilizing constraints by mutation in the DRY motif leads to the formation of active GPCR conformations, but concomitantly to GPCR instability.
authors.affiliation: Leiden/Amsterdam Center for Drug Research, Medicinal Chemistry, Division of Chemistry, Faculty of Sciences, Vrije Universiteit, De Boelelaan, HV Amsterdam, The Netherlands.
authors.forename: A E
authors.initials: AE
authors.lastname: Alewijnse
authors.forename: H
authors.initials: H
authors.lastname: Timmerman
authors.forename: E H
authors.initials: EH
authors.lastname: Jacobs
authors.forename: M J
authors.initials: MJ
authors.lastname: Smit
authors.forename: E
authors.initials: E
authors.lastname: Roovers
authors.forename: S
authors.initials: S
authors.lastname: Cotecchia
authors.forename: R
authors.initials: R
authors.lastname: Leurs
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D006224:Cricetinae; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011899:Ranitidine / Q000494:pharmacology; D018340:Receptors, Adrenergic, alpha-1 / Q000235:genetics / Q000378:metabolism; D011970:Receptors, Histamine H2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D008024:Ligands; D018340:Receptors, Adrenergic, alpha-1; D011970:Receptors, Histamine H2; D011899:Ranitidine; D001120:Arginine