Mutation of Asn-391 within the conserved NPXXY motif of the cholecystokinin B receptor abolishes Gq protein activation without affecting its association with the receptor.
Among the most conserved regions in the G-protein-coupled receptors is the (N/D)PX(2-3)Y motif of the seventh transmembrane domain (X represents any amino 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 adenylyl 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 mutation on binding, signaling, and G-proteins coupling after expression of the mutated receptor in COS cells. The mutated receptor displayed similar expression levels and high affinity CCK binding compared with the wild type CCKBR. However, unlike the wild type CCKBR, the mutated receptor was completely unable to mediate activation of either phospholipase C and protein kinase C-dependent and -independent mitogen-activated protein kinase pathways, indicating an essential role of Asn-391 in CCKBR signaling. Coimmunoprecipitation experiments allowed us to show that the inactive mutant retains an intact capacity to form stable complexes with G(q)alpha subunits in response to CCK. These results indicate that the formation of high affinity CCK-receptor-G(q) protein complexes is not sufficient to activate G(q) and suggest that Asn-391 is specifically involved in G(q) proteins activation.
Galés
C
C
INSERM U 531, Institut Louis Bugnard, CHU Rangueil, Bat L3, 31403 Toulouse Cedex, France.
Kowalski-Chauvel
A
A
Dufour
M N
MN
Seva
C
C
Moroder
L
L
Pradayrol
L
L
Vaysse
N
N
Fourmy
D
D
Silvente-Poirot
S
S
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001216:Asparagine; D001224:Aspartic Acid; D001665:Binding Sites; D019556:COS Cells; D002766:Cholecystokinin / Q000494:pharmacology; D017124:Conserved Sequence; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D043887:Receptor, Cholecystokinin B; D011949:Receptors, Cholecystokinin / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism
D007295:Inositol Phosphates; D010446:Peptide Fragments; D043887:Receptor, Cholecystokinin B; D011949:Receptors, Cholecystokinin; D011994:Recombinant Proteins; C053399:cholecystokinin 9; D001224:Aspartic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D001216:Asparagine; D002766:Cholecystokinin; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D043802:GTP-Binding Protein alpha Subunits, Gq-G11