216 The Bile Acid Receptor GpBAR1 is Regulated by β-Arrestin-Independent Mechanisms and Transactivates the Epidermal Growth Factor Receptor Within Plasma Membrane Microdomains

ation was assessed by metabolic labeling cells with [32P]ortho-phosphate followed by immunoprecipitation (IP) with receptor-specific antibodies. Protein-protein interactions were assessed by IP followed by immunoblotting. All experiments were repeated a minimum of three times. Results. 1) PMA-sensitive PKCs regulate both basal and G17-induced CCK2R densensitization since: a) pretreatment with PMA (100nM, 10 min) stimulated basal phosphorylation of CCK2R, reduced the amplitude of the initial G17-induced increase in [Ca]i compared to untreated control cells (heterologus desensitization), and enhanced receptor desensitization to repeated G17 stimulation (homologus desensitization); b) siRNA-mediated KD of either PMA-sensitive PKCα, -β, -δ, or -θ inhibited CCK2R desensitization. 2) RKIP and GRK2 regulate CCK2 receptor desensitization in a PKC-dependent manner since: a) siRNA-mediated KD of GRK2 expression inhibited CCK2R desensitization whereas KD of endogenous RKIP enhanced receptor desensitization; b) PMA increased the co-IP of GRK2 with RKIP, but not with S153A-RKIP; c) overexpression of S153A-RKIP increased G17induced CCK2R desensitization compared to cells expressing control expression vector. Conclusion. PMA-sensitive PKCs regulate CCK2R desensitization directly by affecting the basal level of CCK2 phosphorylation and indirectly through RKIP-mediated inhibition of GRK2. These data suggest that CCK2R desensitizationmay be inhibitedwhen PKC expression/ activity are reduced, such as during colorectal carcinogenesis.