Functional characterization of a series of mutant G protein α(q) subunits displaying promiscuous receptor coupling properties

Abstract

The N termini of two G protein α subunits, α(q) and α11, differ from those of other α subunits in that they display a unique, highly conserved six-amino acid extension (MTLESI(M)). We recently showed that an α(q) deletion mutant lacking these six amino acids (in contrast to wild type α(q)) was able to couple to several different G(s)- and G(i/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 α(q/11) are critical for constraining the receptor coupling selectivity of these subunits, this region of α(q) was subjected to systematic deletion and alanine scanning mutagenesis. All mutant α(q) constructs (or wild type α(q) as a control) were coexpressed (in COS-7 cells) with the m2 muscarinic or the D2 dopamine receptors, two prototypical G(i/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 α(q)) gained the ability to productively interact with the two G(i/o)-linked receptors. Similar results were obtained when we examined the ability of selected mutant α(q) subunits to couple to the G(s)-coupled β2-adrenergic receptor. Additional experiments indicated that the functional promiscuity displayed by all investigated mutant α(q) constructs was not due to overexpression (as compared with wild type α(q)), 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 α(q/11) subunits forms a tightly folded protein subdomain that is critical for regulating the receptor coupling selectivity of these subunits.