Role of Receptor and Protein Kinase C Activation in the Internalization of the Gastrin-Releasing Peptide Receptor
- Department of Integrative Biology and Pharmacology, University of Texas Medical School, Houston, Texas 77225
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 (Ki = 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 (KD = 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
-
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.
- Abbreviations:
- GPCR
- G protein-coupled receptor
- Bn
- bombesin
- PA
- propylamide
- GRP
- gastrin-releasing peptide
- GRP-R
- gastrin-releasing peptide receptor
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- HBSS
- HEPES-buffered salt solution, pH 7.2
- TFA
- trifluoroacetic acid
- PMA
- phorbol-12-myristate-13-acetate
- PKC
- protein kinase C
- GRK
- G protein-coupled receptor kinase
- PLC
- phospholipase C
- Gpp(NH)p
- guanosine-5′-(β,γ-imido)triphosphate
-
- Received March 30, 1998.
- Accepted July 30, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
