CONSTRUCTION OF A NOVEL BIFUNCTIONAL BIOGENIC-AMINE RECEPTOR BY 2 POINT MUTATIONS OF THE H2-HISTAMINE RECEPTOR

Background: H2-histamine receptors mediate a wide range of physiologic al functions extending from stimulation of gastric acid secretion to i nduction of human promyelocyte differentiation. We have previously clo ned the H2-histamine receptor gene and noted that only three amino aci ds on the receptor were sufficient to define its specificity and selec tivity. Despite only modest overall amino acid homology (34% amino add identity and 57.5% similarity) between the H2-histamine receptor and the receptor for another monoamine, the beta 2-adrenergic receptor, th ere is remarkable similarity at their critical ligand binding sites. W e hypothesized that, if the specificity and selectivity of both recept ors are invested in just three amino acids, it should be possible to c onvert one of the receptors into one that recognizes the ligand of the other by simple mutations at only one or two sites. Material and Meth ods: We explored the effect of two single mutations in the fifth trans membrane domain of the H2-histamine receptor, which encompasses the si tes that determine H2 selectivity. The canine H2 receptor gene was mut ated at Asp(186) and Gly(187) (Asp(186) to Ala(186) and Gly(187) to Se r(187)) by oligonuceotide directed mutagenesis. The coding region of b oth the wild-type and mutated H2 receptors was subcloned into the euka ryotic expression vector, CMVneo, and stably transfected into Hepa cel ls and L cells. The biological activity of histamine and epinephrine o n the expressed receptor was examined by measurement of cellular cAMP production and inositol trisphosphate formation. Results: Hepa cells t ransfected with the Ala(186)-Ser(187) mutant H2 receptor demonstrated a 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 pro panolol and cimetidine. Epinephrine also induced IP3 generation in the same cells, a biological response that is characteristic of activatio n of the wild-type H2 but not of the P-adrenergic receptor. L cells tr ansfected with the Ala(186)-Ser(187) mutant H2 receptor also responded to epinephrine in a cimetidine and propranolol inhibitable manner. Co nclusions: We converted the H2-histamine receptor into a bifunctional one that has characteristics of both histamine and adrenergic receptor s bp two simple mutations. These results support the hypothesis that l igand specificity is determined by only a few key points on a receptor regardless of the structure of the remainder of the molecule. Our stu dies have important implications on the design of pharmacological agen ts targeted for action at physiological receptors.