Received for publication April 8, 2004.
Revised August 26, 2004.
Accepted for publication September 10, 2004.

Alterations in Receptor Expression or Agonist Concentration Change the Pathways Gastrin-Releasing Peptide Receptor Use to Regulate ERK

Abstract

G protein-coupled receptors (GPCRs) activate extracellular signal-regulated kinases (ERKs) via different pathways in different cell types. In this study we demonstrate that gastrin-releasing peptide receptor (GRPr) regulates ERK through multiple pathways in a single cell type depending upon receptor expression and agonist concentration. We examined stably transfected Balb/c 3T3 fibroblasts expressing GRPr constructs at different levels and treated the cells with several concentrations of bombesin (BN, a GRPr agonist) to activate a variable number of GRPr per cell. GRPr induced two waves of ERK activation and one wave of ERK inhibition. One wave of activation required an intact GRPr carboxyl terminal domain (CTD). It peaked 6 minutes after addition of high BN concentration ([BN]) in cells with high GRPr expression. Another wave of activation was CTD-independent. It peaked 2-4 min after BN addition in cells when [BN] and/or GRPr expression were lower. The early wave of ERK activation was more sensitive than the later one to pretreatment with GF109203X (a PKC inhibitor) or hypertonic sucrose. Since these two waves of activation differ in time course, dose-response curve, requirement for GRPr CTD, and sensitivity to inhibitors, they result from different signaling pathways. A third pathway in these cells inhibited ERK phosphorylation 2 min after addition of high [BN] in cells with high GRPr expression. Furthermore, a GRPr-expressing human duodenal cancer cell line showed differential sensitivity to GF109203X throughout BN-induced ERK activation, indicating that GRPr may activate ERK via multiple pathways in cells expressing endogenous GRPr.

Key words: Bombesin, Gq/11 family, Protein Kinase C, Protein tyr Phosphatases, Sequestration/Internalization, GRKs, barrestins, Phosphorylation/Dephosphorylation, MAP Kinase, Func. analysis receptor/ion channel mutants

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