Proline-rich Motifs in the Parathyroid Hormone (PTH)/PTH-related Protein Receptor C Terminus Mediate Scaffolding of c-Src with β-Arrestin2 for ERK1/2 Activation

Alexandre Rey,Danielle Manen,Joseph Caverzasio,Serge L Ferrari,René Rizzoli

doi:10.1074/jbc.m606762200

Alexandre Rey, Danielle Manen + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m606762200

Copy DOI

Abstract

Parathyroid hormone (PTH) stimulates ERK1/2 through both G-protein signaling and beta-arrestin2-mediated internalization. Beta-arrestin may serve as a scaffold for c-Src. However, the molecular mechanisms for ERK1/2 activation by PTH remain unclear. By using a targeted mutagenesis approach, we investigated the PTH/PTH-related protein receptor (PTH1R) structural determinants for ERK1/2 activation and transcriptional activity in HEK-293 cells. First, ERK1/2 activation was inhibited by PTH1R mutations that specifically abrogate G(q)-protein kinase C signaling without a decrease in cAMP-protein kinase A. Second, PTH1R C-terminal mutations and/or deletions that prevent interaction with beta-arrestin inhibited ERK1/2 activation. Similar results were obtained in HEK-293 cells co-expressing wild-type PTH1R and a dominant-negative beta-arrestin2. Third, the c-Src inhibitor PP2 and a kinase-dead c-SrcK295M mutant co-expressed with wild-type PTH1R both inhibited ERK1/2 activation. Furthermore, c-Src co-precipitated with both PTH1R and beta-arrestin2 in response to PTH. Deleting the PTH1R-proximal C terminus abolished these interactions. However, the need for receptor interaction with beta-arrestin to co-precipitate Src and activate ERK1/2 was obviated by expressing a constitutively active c-SrcY527A mutant, suggesting direct binding of activated Src to PTH1R. Subsequently, we identified and mutated to alanine four proline-rich motifs in the PTH1R distal C terminus, which resulted in loss of both c-Src and arrestin co-precipitation and significantly decreased ERK1/2 activation. These data delineate the multiple PTH1R structural determinants for ERK1/2 activation and newly identify a unique mechanism involving proline-rich motifs in the receptor C terminus for reciprocal scaffolding of c-Src and beta-arrestin2 with a class II G-protein-coupled receptor.

Highlights

(GPCR) agonists, including peptidic hormones, regulate crucial biological processes such as cell growth and differentiation [1]
Recent studies on the ␤2-adrenergic receptor and the angiotensin type 1A receptor showed that ERK1/2 could be independently activated by the classical Gs and Gq signaling cascades and/or through receptor internalization with ␤-arrestins [2,3,4]. ␤-Arrestin 1 and 2 are cytosolic proteins that interact with GPCRs that have been phosphorylated by a variety of kinases such as protein kinase A (PKA), protein kinase C (PKC), and G-protein-coupled receptor kinases (GRKs) [2, 5]
By using an extensive mutagenesis approach, we identify a unique mechanism for ERK1/2 activation by Parathyroid hormone (PTH) that engages scaffolding of type; dn, dominant negative; PKC, protein kinase C; PKA, protein kinase A; GRK, G-protein-coupled receptor kinase; SH3, Src homology 3; SRE, serumresponsive element; EGFR, epidermal growth factor receptor; FCS, fetal calf serum

Summary

Introduction

(GPCR) agonists, including peptidic hormones, regulate crucial biological processes such as cell growth and differentiation [1]. PTH-stimulated cAMP Signaling by WT and Mutant PTH1R—To perform the structure-function analyses of PTH1R described in detail below, we generated a number of point mutations and/or deletions in the receptor second intracellular loop (IC2) and C terminus, as shown, A and B.

Results

Conclusion