title: Functional characterization of a series of mutant G protein alphaq subunits displaying promiscuous receptor coupling properties.
abstract: The N termini of two G protein alpha subunits, alphaq and alpha11, differ from those of other alpha subunits in that they display a unique, highly conserved six-amino acid extension (MTLESI(M)). We recently showed that an alphaq deletion mutant lacking these six amino acids (in contrast to wild type alphaq) was able to couple to several different Gs- and Gi/o-coupled receptors, apparently due to promiscuous receptor/G protein coupling (Kostenis, E., Degtyarev, M. Y., Conklin, B. R., and Wess, J. (1997) J. Biol. Chem. 272, 19107-19110). To study which specific amino acids within the N-terminal segment of alphaq/11 are critical for constraining the receptor coupling selectivity of these subunits, this region of alphaq was subjected to systematic deletion and alanine scanning mutagenesis. All mutant alphaq constructs (or wild type alphaq as a control) were coexpressed (in COS-7 cells) with the m2 muscarinic or the D2 dopamine receptors, two prototypical Gi/o-coupled receptors, and ligand-induced increases in inositol phosphate production were determined as a measure of G protein activation. Surprisingly, all 14 mutant G proteins studied (but not wild type alphaq) gained the ability to productively interact with the two Gi/o-linked receptors. Similar results were obtained when we examined the ability of selected mutant alphaq subunits to couple to the Gs-coupled beta2-adrenergic receptor. Additional experiments indicated that the functional promiscuity displayed by all investigated mutant alphaq constructs was not due to overexpression (as compared with wild type alphaq), lack of palmitoylation, or initiation of translation at a downstream ATG codon (codon seven). These data are consistent with the notion that the six-amino acid extension characteristic for alphaq/11 subunits forms a tightly folded protein subdomain that is critical for regulating the receptor coupling selectivity of these subunits.
authors.affiliation: Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: E
authors.initials: E
authors.lastname: Kostenis
authors.forename: F Y
authors.initials: FY
authors.lastname: Zeng
authors.forename: J
authors.initials: J
authors.lastname: Wess
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D014176:Protein Biosynthesis; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D017384:Sequence Deletion
chemical_list: D018343:Receptors, Adrenergic, beta-2; D019204:GTP-Binding Proteins