Several domains of G protein alpha subunits are implicated in the control of receptor-G protein coupling specificity. Among these are the extreme N-and C-termini, the alpha4/beta6-loops, and the loop linking the N-terminal alpha-helix to the beta1-strand of the ras-like domain. In this study, we illustrate that single-point mutations of a highly conserved glycine residue within the linker I region of the Galpha(q) subunit confers upon the mutant Galpha(q) the ability to be activated by Galpha(i)- and Galpha(s) -coupled receptors, as evidenced by guanosine 5'-O-(3-[(35)S]thio)triphosphate binding and inositol phosphate turnover assays. The mutations did not affect expression of Galpha(q) proteins nor their ability to stimulate phospholipase Cbeta. It is noteworthy that both mutant and wild-type Galpha(q) proteins are indistinguishable in their ability to reconstitute a functional Gq-PLCbeta-calcium signaling pathway when cotransfected with the Galpha(q)-coupled neurokinin 1 or muscarinic M3 receptor into mouse embryonic fibroblasts derived from Galpha(q/11) knockout mice. On a three-dimensional model of the receptor-G protein complex, the highly conserved linker I region connecting the helical and the GTPase domain of the Galpha protein is inaccessible to the intracellular surface of the receptors. Our data indicate that receptor-G protein coupling specificity is not exclusively governed by direct receptor-G protein interaction and that it even bypasses the requirement of the extreme C terminus of Galpha, a well accepted receptor recognition domain, suggesting a novel allosteric mechanism for G protein-coupled receptor-G protein selectivity.