Inhibition of GDP/GTP exchange on Gα subunits by proteins containing G-protein regulatory motifs

A novel G alpha binding consensus sequence, termed G-protein regulatory (CiPR) or GoLoco motif, has been identified in a growing number of proteins, which are thought to modulate G-protein signaling. Alternative roles of GPR proteins: as nucleotide exchange factors or as GDP dissociation inhibitors for G alpha have been proposed. We investigated the modulation of the GDP/GTP exchange of Gi alpha (1), Go alpha, and Gs alpha by three proteins containing CPR motifs (GPR proteins), LGN-585-642, Pcp2, and RapIGAPII-23-131, to elucidate the mechanisms of GPR protein function. The GPR proteins displayed similar patterns of interaction with Gi alpha with the following order of affinities: Gi alpha (1)GDP much greater than Gi alpha (1)GDPAlF(4)(-) greater than or equal to Gi alpha (1)-GTP gammaS. No detectable binding of the GPR proteins to Gs alpha was observed, LGN-585-642, Pcp2. and RapIGAPII-23-131 inhibited the rates of spontaneous GTP gammaS binding and blocked GDP release from Ci alpha (1) and Go alpha. The inhibitory effects of the GPR proteins on Gi alpha (1) were significantly more potent, indicating that Gi might be a preferred target for these modulators. Our results suggest that GPR proteins are potent GDP dissociation inhibitors for Ci alpha -like G alpha subunits in vitro, and in this capacity they may inhibit GPCR/ Gi protein signaling in vivo.