Dynamic interaction of human vasopressin/oxytocin receptor subtypes with Gprotein-coupled receptor kinases and protein kinase C after agonist stimulation

Examination of the structure of [Arg(8)]-vasopressin receptors (AVPRs) and oxytocin receptors (OTRs) suggests that G protein-coupled receptor kinases (GBKs) and protein kinase C (PKC) are involved in their signal transduction . To explore the physical association of AVPRs and OTRs with GRKs and PKC, wild types and mutated forms of these receptor subtypes were stably express ed as green fluorescent protein fusion proteins and analyzed by fluorescenc e, immunoprecipitation, and immunoblotting. Addition of a C-terminal GFP ta g did not interfere with ligand binding, internalization, and signal transd uction, After agonist stimulation, PKC dissociated from the V1R, did not as sociate with the V2R, but associated with the V3R and the OTR. After AVP st imulation, only GRK5 briefly associated with AVPRs following a time course that varied with the receptor subtype. No GRK associated with the OTR, Exch anging the V1R and V2R. C termini altered the time course of PKC and GRK5 a ssociation. Deletion of the V1R C terminus resulted in no PKC association a nd a ligand-independent sustained association of GRK5 with the receptor. De letion of the GRK motif prevented association and reduced receptor phosphor ylation, Thus, agonist stimulation of AVP/OT receptors leads to receptor su btype-specific interactions with GRK and PKC through specific motifs presen t in the C termini of the receptors.