Proline-rich motifs in the parathyroid hormone (PTH)/PTH-related protein receptor C terminus mediate scaffolding of c-Src with beta-arrestin2 for ERK1/2 activation.
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.
Rey
Alexandre
A
Service of Bone Diseases, Department of Rehabilitation and Geriatrics, University Hospital, 1211 Geneva 14, Switzerland.
Manen
Danielle
D
Rizzoli
René
R
Caverzasio
Joseph
J
Ferrari
Serge L
SL
D000595:Amino Acid Sequence; D019390:Arrestins / Q000378:metabolism*; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D020935:MAP Kinase Signaling System; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D055232:Proline-Rich Protein Domains; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D015398:Signal Transduction; D000071557:beta-Arrestins
D019390:Arrestins; D010455:Peptides; D044168:Receptor, Parathyroid Hormone, Type 1; D000071557:beta-Arrestins; D000242:Cyclic AMP; D016392:Proto-Oncogene Proteins pp60(c-src); D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3