AGONIST BINDING AND PROTEIN-KINASE-C ACTIVATION STIMULATE PHOSPHORYLATION OF THE GASTRIN-RELEASING PEPTIDE RECEPTOR AT DISTINCT SITES

Gastrin-releasing peptide and other bombesin-like peptides stimulate s ecretion, cell proliferation, and smooth muscle contraction via a fami ly of G protein-coupled receptors that activate phospholipase C. Secon d messenger formation by one of these receptors, called BR1, is rapidl y desensitized after treatment of cells with either agonists or the pr otein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA). T o determine whether receptor phosphorylation was involved in BR1 desen sitization, we generated antibodies to a peptide corresponding to a un ique sequence within the COOH terminus of this receptor. One antibody (BR1-517) immunoprecipitated 60% of the solubilized [I-125-Ty(4)]bombe sin/receptor complex prepared from either Swiss 3T3 fibroblasts or CHO -K1 cells transfected to express high levels of mouse BR1 (CHO-mBR1). Furthermore, immunoprecipitation of photoaffinity-labeled receptors yi elded the expected 87-kDa radiolabeled band on gel electrophoresis. Ph osphorylation of this immunoprecipitated receptor protein was markedly stimulated when [P-32]orthophosphate-labeled Swiss 3T3 cells or CHO-m BR1 cells were treated with 100 nM bombesin for 5 min, (PO4)-P-32 inco rporation into immunoprecipitated receptor was detectable after 2 min and maximal after 15 min of bombesin treatment. Phosphoamino acid anal ysis showed P-32 labeling of serine and threonine but not tyrosine res idues. Pretreatment of CHO-mBR1 cells with 100 nM TPA for 30 min also desensitized bombesin stimulation of inositol-1,4,5-trisphosphate form ation. However, TPA did not increase (PO4)-P-32 incorporation into the immunoprecipitated receptor, although protein kinase C inhibition pot entiated bombesin-induced receptor phosphorylation. Subsequent studies showed that TPA did stimulate receptor phosphorylation, but the antib ody did not recognize this phosphorylated state of the receptor. Thus, TPA decreased the efficiency of receptor immunoprecipitation, and sub sequent incubation of receptor with alkaline phosphatase reversed this TPA inhibition, The differential specificity of the antibody for vari ous phosphorylated forms of BR1 demonstrates that agonist-induced and TPA-induced phosphorylations of the receptor occur at distinct sites.