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 secretion, cell proliferation, and smooth muscle contraction via a family of G protein-coupled receptors that activate phospholipase C. Second messenger formation by one of these receptors, called BR1, is rapidly desensitized after treatment of cells with either agonists or the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA). To determine whether receptor phosphorylation was involved in BR1 desensitization, we generated antibodies to a peptide corresponding to a unique sequence within the COOH terminus of this receptor. One antibody (BR1-517) immunoprecipitated 60% of the solubilized [I-125-Ty(4)]bombesin/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 yielded the expected 87-kDa radiolabeled band on gel electrophoresis. Phosphorylation of this immunoprecipitated receptor protein was markedly stimulated when [P-32]orthophosphate-labeled Swiss 3T3 cells or CHO-mBR1 cells were treated with 100 nM bombesin for 5 min, (PO4)-P-32 incorporation into immunoprecipitated receptor was detectable after 2 min and maximal after 15 min of bombesin treatment. Phosphoamino acid analysis showed P-32 labeling of serine and threonine but not tyrosine residues. Pretreatment of CHO-mBR1 cells with 100 nM TPA for 30 min also desensitized bombesin stimulation of inositol-1,4,5-trisphosphate formation. However, TPA did not increase (PO4)-P-32 incorporation into the immunoprecipitated receptor, although protein kinase C inhibition potentiated bombesin-induced receptor phosphorylation. Subsequent studies showed that TPA did stimulate receptor phosphorylation, but the antibody did not recognize this phosphorylated state of the receptor. Thus, TPA decreased the efficiency of receptor immunoprecipitation, and subsequent incubation of receptor with alkaline phosphatase reversed this TPA inhibition, The differential specificity of the antibody for various phosphorylated forms of BR1 demonstrates that agonist-induced and TPA-induced phosphorylations of the receptor occur at distinct sites.