ROLE OF RECEPTOR AND PROTEIN-KINASE-C ACTIVATION IN THE INTERNALIZATION OF THE GASTRIN-RELEASING PEPTIDE RECEPTOR

The mechanisms regulating receptor internalization are not well unders tood and vary among different G protein-coupled receptors. The bombesi n (Bn)/gastrin-releasing peptide receptor GRP-R, which is coupled to p hospholipase C via the G(q) family of transducing proteins, is interna lized rapidly after Bn binding. Agonist stimulation leads to rapid rec eptor phosphorylation, as does activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA), However, agonist- and PMA-indu ced phosphorylation occur at different receptor sites. Here, we examin ed the role of PKC in GRP-R internalization after agonist and antagoni st binding. We synthesized [D-Tyr(6)]Bn(6-13)propylamide ([D-Tyr(6)]Bn (6-13)PA) and found that it potently inhibited Bn-stimulated insulin r elease and [I-125-Tyr(4)]Bn binding (K-i = 4.72 nM) in the HIT-T15 pan creatic cell line. The radiolabeled antagonist peptide, [I-125-D-Tyr(6 )]Bn(6-13)PA, bound with high affinity (K-D = 0.29 nM at 4 degrees) to a single class of receptor sites, and competition binding studies exh ibited the analog specificity expected for the GRP-R subtype. Although the agonist [I-125-Tyr(4)]Bn was internalized rapidly at 37 degrees a nd subsequently degraded, [I-125-D-Tyr(6)]Bn(6-13)PA was not internali zed and was released into the medium mainly as intact peptide. The lys osomal inhibitor chloroquine (200 mu M) increased the intracellular ac cumulation of [I-125-Tyr(4)]Bn but had no effect on the subcellular di stribution of [I-125-D-Tyr(6)]Bn(6-13)PA. Consistent with these observ ations, the treatment of cells with 100 nM Bn at 37 degrees reduced ce ll surface receptors within minutes, whereas [D-Tyr(6)]Bn(6-13)PA had no effect. The addition of PMA did not induce the internalization of a ntagonist-occupied receptors, but pharmacological inhibition of PKC de creased the rate of agonist-induced receptor internalization. These re sults therefore demonstrate that although PKC contributes to agonist-i nduced internalization of the GRP-R, it does not elicit receptor inter nalization of the antagonist-occupied receptor.