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 Gq 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.
Footnotes
- Received March 30, 1998.
- Accepted July 30, 1998.
-
Send reprint requests to: Agnes Schonbrunn, Ph.D., Department of Integrative Biology and Pharmacology, University of Texas Medical School, P.O. Box 20708, Houston, TX 77225. E-mail:aschonb{at}farmr1.med.uth.tmc.edu
-
↵1 Current affiliation: Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030.
-
↵2 Current affiliation: McGill University, Lady Davis Institute, Montreal, Quebec, Canada H3T 1E2.
-
This investigation was supported by the Texas Advanced Technology Program (Grant 1823). B.Y.W. is the recipient of a Pharmaceutical Manufacturers Association Foundation Advanced Predoctoral Fellowship. S.B.D. was supported by a postdoctoral fellowship from the Fonds de la Recherche on Sante du Quebec.
- The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|