Abstract

The mechanisms regulating receptor internalization are not well understood and vary among different G protein-coupled receptors. The bombesin (Bn)/gastrin-releasing peptide receptor GRP-R, which is coupled to phospholipase C via the G_q family of transducing proteins, is internalized rapidly after Bn binding. Agonist stimulation leads to rapid receptor phosphorylation, as does activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA). However, agonist- and PMA-induced phosphorylation occur at different receptor sites. Here, we examined the role of PKC in GRP-R internalization after agonist and antagonist binding. We synthesized [d-Tyr⁶]Bn(6–13)propylamide ([d-Tyr⁶]Bn(6–13)PA) and found that it potently inhibited Bn-stimulated insulin release and [¹²⁵I-Tyr⁴]Bn binding (K _i = 4.72 nm) in the HIT-T15 pancreatic cell line. The radiolabeled antagonist peptide, [¹²⁵I-d-Tyr⁶]Bn(6–13)PA, bound with high affinity (K _D = 0.29 nm at 4°) to a single class of receptor sites, and competition binding studies exhibited the analog specificity expected for the GRP-R subtype. Although the agonist [¹²⁵I-Tyr⁴]Bn was internalized rapidly at 37° and subsequently degraded, [¹²⁵I-d-Tyr⁶]Bn(6–13)PA was not internalized and was released into the medium mainly as intact peptide. The lysosomal inhibitor chloroquine (200 μm) increased the intracellular accumulation of [¹²⁵I-Tyr⁴]Bn but had no effect on the subcellular distribution of [¹²⁵I-d-Tyr⁶]Bn(6–13)PA. Consistent with these observations, the treatment of cells with 100 nm Bn at 37° reduced cell surface receptors within minutes, whereas [d-Tyr⁶]Bn(6–13)PA had no effect. The addition of PMA did not induce the internalization of antagonist-occupied receptors, but pharmacological inhibition of PKC decreased the rate of agonist-induced receptor internalization. These results therefore demonstrate that although PKC contributes to agonist-induced internalization of the GRP-R, it does not elicit receptor internalization of the antagonist-occupied receptor.