GENETIC-ANALYSIS OF RECEPTOR-G-ALPHA(Q) COUPLING SELECTIVITY

Many different G protein-linked receptors are preferentially coupled t o G proteins of the G(q/11) family, To elucidate the molecular basis u nderlying this selectivity, different G(q/11)-coupled receptors (m3 mu scarinic, V1a vasopressin, and gastrin-releasing peptide receptor) wer e coexpressed (in COS-7 cells) with mutant alpha(s) subunits in which residues present at the C terminus of alpha(s) were replaced with the corresponding alpha(q/11), residues, Remarkably, whereas none of the r eceptors was able to interact with wild type alpha(s) to a significant extent, all three receptors gained the ability to productively couple to a mutant alpha(s) subunit containing a single Glu --> Asn point mu tation at position -3, Moreover, the m3 muscarinic and the Via vasopre ssin receptors but not the GRP receptor also gained the ability to int eract with a mutant alpha(s) subunit containing a single Gln --> Glu p oint mutation at position -5, indicating that the alpha(q/11) residues present in these mutant G protein constructs play key roles in determ ining the selectivity of receptor recognition. To identify the site(s) on G(q/11)-coupled receptors that can functionally interact with the C terminus of alpha(q/11) subunits, we next analyzed the ability of a series of hybrid m2/m3 muscarinic receptors to interact with a mutant alpha(s) subunit (sq5) in which the last five amino acids of alpha(s) were replaced with the corresponding alpha(q/11) sequence, Similar to the wild type m2 and m3 muscarinic receptors, none of the investigated hybrid receptors was able to efficiently interact with wild type alph a(s). Interestingly, however, three mutant m2 receptors in which diffe rent segments of the second and third intracellular loops were replace d with the corresponding m3 receptor sequences were identified, which, in contrast to the G(i/o)-coupled wild type m2 receptor, gained the a bility to efficiently activate the sq5 subunit, This observation sugge sts that multiple intracellular receptor domains form a binding pocket for the C terminus of G protein alpha(q/11) subunits.