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Titolo:
CONSTRUCTION OF A NOVEL BIFUNCTIONAL BIOGENIC-AMINE RECEPTOR BY 2 POINT MUTATIONS OF THE H2-HISTAMINE RECEPTOR
Autore:
DELVALLE J; GANTZ I; WANG LD; GUO YJ; MUNZERT G; TASHIRO T; KONDA Y; YAMADA T;
Indirizzi:
6520 MSRBI ANN ARBOR MI 48109 UNIV MICHIGAN,MED CTR,DEPT INTERNAL MED ANN ARBOR MI 48109 UNIV MICHIGAN,MED CTR,DEPT SURG ANN ARBOR MI 48109 UNIV MICHIGAN,MED CTR,DEPT PHYSIOL ANN ARBOR MI 48109
Titolo Testata:
Molecular medicine
fascicolo: 3, volume: 1, anno: 1995,
pagine: 280 - 286
SICI:
1076-1551(1995)1:3<280:COANBB>2.0.ZU;2-S
Fonte:
ISI
Lingua:
ENG
Soggetto:
BETA-ADRENERGIC-RECEPTOR; MOLECULAR-BASIS; SPECIES SELECTIVITY; SUBSTANCE-P; ACID; IDENTIFICATION; ANTAGONISTS; BINDING; HISTAMINE-H2-RECEPTOR; ANTAGONIST-CP-96,345;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Science Citation Index Expanded
Citazioni:
17
Recensione:
Indirizzi per estratti:
Citazione:
J. Delvalle et al., "CONSTRUCTION OF A NOVEL BIFUNCTIONAL BIOGENIC-AMINE RECEPTOR BY 2 POINT MUTATIONS OF THE H2-HISTAMINE RECEPTOR", Molecular medicine, 1(3), 1995, pp. 280-286

Abstract

Background: H2-histamine receptors mediate a wide range of physiological functions extending from stimulation of gastric acid secretion to induction of human promyelocyte differentiation. We have previously cloned the H2-histamine receptor gene and noted that only three amino acids on the receptor were sufficient to define its specificity and selectivity. Despite only modest overall amino acid homology (34% amino add identity and 57.5% similarity) between the H2-histamine receptor andthe receptor for another monoamine, the beta 2-adrenergic receptor, there is remarkable similarity at their critical ligand binding sites. We hypothesized that, if the specificity and selectivity of both receptors are invested in just three amino acids, it should be possible to convert one of the receptors into one that recognizes the ligand of the other by simple mutations at only one or two sites. Material and Methods: We explored the effect of two single mutations in the fifth transmembrane domain of the H2-histamine receptor, which encompasses the sites that determine H2 selectivity. The canine H2 receptor gene was mutated at Asp(186) and Gly(187) (Asp(186) to Ala(186) and Gly(187) to Ser(187)) by oligonuceotide directed mutagenesis. The coding region of both the wild-type and mutated H2 receptors was subcloned into the eukaryotic expression vector, CMVneo, and stably transfected into Hepa cells and L cells. The biological activity of histamine and epinephrine on the expressed receptor was examined by measurement of cellular cAMPproduction and inositol trisphosphate formation. Results: Hepa cells transfected with the Ala(186)-Ser(187) mutant H2 receptor demonstrateda biphasic rise in cAMP in response to epinephrine with an early phase (ED(50) approximate to 10(-11) M) that could be inhibited by both propanolol and cimetidine. Epinephrine also induced IP3 generation in the same cells, a biological response that is characteristic of activation of the wild-type H2 but not of the P-adrenergic receptor. L cells transfected with the Ala(186)-Ser(187) mutant H2 receptor also responded to epinephrine in a cimetidine and propranolol inhibitable manner. Conclusions: We converted the H2-histamine receptor into a bifunctionalone that has characteristics of both histamine and adrenergic receptors bp two simple mutations. These results support the hypothesis that ligand specificity is determined by only a few key points on a receptor regardless of the structure of the remainder of the molecule. Our studies have important implications on the design of pharmacological agents targeted for action at physiological receptors.

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Documento generato il 23/09/20 alle ore 12:55:54