Mutation of Asn-391 within the conserved NPXXY motif of the cholecystokinin B receptor abolishes G(q) protein activation without affecting its association with the receptor

Among the most conserved regions in the G-protein-coupled receptors is the (N/D)PX2-3Y motif of the seventh transmembrane domain (X represents any ami no acid). The mutation of the Asn/Asp residue of this motif in different G- protein-coupled receptors was shown to affect the activation of either aden ylyl cyclase or phospholipase C. We have mutated the Asn residue (Asn-391) of the NPXXY motif in the CCKBR to Ala and determined the effects of the mu tation on binding, signaling, and G-proteins coupling after expression of t he mutated receptor in COS cells. The mutated receptor displayed similar ex pression levels and high affinity CCK binding compared with the wild type C CKBR, However, unlike the wild type CCKBR, the mutated receptor was complet ely unable to mediate activation of either phospholipase C and protein kina se C-dependent and -independent mitogen-activated protein kinase pathways, indicating an essential role of Asn-391 in CCKBR signaling. Coimmunoprecipi tation experiments allowed us to show that the inactive mutant retains an i ntact capacity to form stable complexes with G(q)alpha subunits in response to CCK. These results indicate that the formation of high affinity CCK-rec eptor-G(q) protein complexes is not sufficient to activate G(q) and suggest that Asn-391 is specifically involved in G(q),proteins activation.