The effect of RGS12 on PDGFbeta receptor signalling to p42/p44 mitogen activated protein kinase in mammalian cells.

We have previously shown that the PDGFbeta receptor uses a classical GPCR-mediated pathway in order to induce efficient activation of p42/p44 MAPK in response to PDGF. We therefore, considered the possibility that GTPase accelerating proteins (RGS proteins), which regulate GPCR signalling, modulate PDGFbeta receptor-mediated signal transmission. Several lines of evidence were obtained to support functional interaction between the PDGFbeta receptor and RGS12 in HEK 293 and airway smooth muscle cells. Firstly, the over-expression of the RGS12 PDZ/PTB domain N-terminus or RGS12 PTB domain reduced the PDGF-induced activation of p42/p44 MAPK. Secondly, the RGS12 PDZ/PTB domain N-terminus and RGS12 PDZ domain can form a complex with the PDGFbeta receptor. Therefore, the results presented here provide the first evidence to support the concept that the PDZ/PTB domain N-terminus and/or the PTB domain of RGS12 may modulate PDGFbeta receptor signalling. In airway smooth muscle cells, over-expressed recombinant RGS12 and the isolated PDZ/PTB domain N-terminus co-localised with PDGFbeta receptor in cytoplasmic vesicles. To provide additional evidence for a role of the PDZ/PTB domain N-terminus, we used RGS14. RGS14 has the same C-terminal domain architecture of an RGS box, tandem Ras-binding domains (RBDs) and GoLoco motif as RGS12, but lacks the PDZ/PTB domain N-terminus. In this regard, RGS14 exhibited a different sub-cellular distribution compared with RGS12, being diffusely distributed in ASM cells. These findings suggest that RGS12 via its PDZ/PTB domain N-terminus may regulate trafficking of the PDGFbeta receptor in ASM cells.

[1]  T. Palmer,et al.  Tethering of the platelet-derived growth factor beta receptor to G-protein-coupled receptors. A novel platform for integrative signaling by these receptor classes in mammalian cells. , 2001, The Journal of biological chemistry.

[2]  Y. Peterson,et al.  Stabilization of the GDP-bound Conformation of Giα by a Peptide Derived from the G-protein Regulatory Motif of AGS3* , 2000, The Journal of Biological Chemistry.

[3]  C. Betsholtz,et al.  Pericyte‐specific expression of Rgs5: implications for PDGF and EDG receptor signaling during vascular maturation , 2003, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[4]  R. Fisher,et al.  RGS12TS-S Localizes at Nuclear Matrix-Associated Subnuclear Structures and Represses Transcription: Structural Requirements for Subnuclear Targeting and Transcriptional Repression , 2002, Molecular and Cellular Biology.

[5]  J. Sondek,et al.  Structural determinants for GoLoco-induced inhibition of nucleotide release by Gα subunits , 2002, Nature.

[6]  R. Lefkowitz,et al.  Gβγ Subunits Mediate Mitogen-activated Protein Kinase Activation by the Tyrosine Kinase Insulin-like Growth Factor 1 Receptor (*) , 1995, The Journal of Biological Chemistry.

[7]  T. K. Harden,et al.  Application of RGS box proteins to evaluate G-protein selectivity in receptor-promoted signaling. , 2004, Methods in enzymology.

[8]  G. Milligan,et al.  Src-mediated RGS16 Tyrosine Phosphorylation Promotes RGS16 Stability* , 2003, The Journal of Biological Chemistry.

[9]  R. Lefkowitz,et al.  Platelet-Derived Growth Factor Receptor Association with Na+/H+ Exchanger Regulatory Factor Potentiates Receptor Activity , 2000, Molecular and Cellular Biology.

[10]  S. Pyne,et al.  c-Src is involved in regulating signal transmission from PDGFbeta receptor-GPCR(s) complexes in mammalian cells. , 2005, Cellular signalling.

[11]  S. Pyne,et al.  Bradykinin stimulates phospholipase D in primary cultures of guinea-pig tracheal smooth muscle. , 1993, Biochemical pharmacology.

[12]  S. Sprang,et al.  Structure of RGS4 Bound to AlF4 −-Activated Giα1: Stabilization of the Transition State for GTP Hydrolysis , 1997, Cell.

[13]  M. Chao,et al.  GIPC and GAIP form a complex with TrkA: a putative link between G protein and receptor tyrosine kinase pathways. , 2001, Molecular biology of the cell.

[14]  D. Lauffenburger,et al.  The Na+/H+ exchanger regulatory factor stabilizes epidermal growth factor receptors at the cell surface. , 2004, Molecular biology of the cell.

[15]  S. Pyne,et al.  Sphingosine 1-Phosphate and Platelet-derived Growth Factor (PDGF) Act via PDGFβ Receptor-Sphingosine 1-Phosphate Receptor Complexes in Airway Smooth Muscle Cells* , 2003, The Journal of Biological Chemistry.

[16]  S. Pyne,et al.  Platelet-derived-growth-factor stimulation of the p42/p44 mitogen-activated protein kinase pathway in airway smooth muscle: role of pertussis-toxin-sensitive G-proteins, c-Src tyrosine kinases and phosphoinositide 3-kinase. , 1999, The Biochemical journal.

[17]  R. Lefkowitz,et al.  GTPase Activating Specificity of RGS12 and Binding Specificity of an Alternatively Spliced PDZ (PSD-95/Dlg/ZO-1) Domain* , 1998, The Journal of Biological Chemistry.

[18]  Randall J. Kimple,et al.  RGS12 and RGS14 GoLoco Motifs Are GαiInteraction Sites with Guanine Nucleotide Dissociation Inhibitor Activity* , 2001, The Journal of Biological Chemistry.

[19]  J. Kehrl,et al.  The aorta and heart differentially express RGS (regulators of G-protein signalling) proteins that selectively regulate sphingosine 1-phosphate, angiotensin II and endothelin-1 signalling. , 2003, The Biochemical journal.

[20]  Dho Se,et al.  Characterization of four mammalian numb protein isoforms. Identification of cytoplasmic and membrane-associated variants of the phosphotyrosine binding domain. , 1999 .

[21]  S. Muallem,et al.  The N-terminal Domain of RGS4 Confers Receptor-selective Inhibition of G Protein Signaling* , 1998, The Journal of Biological Chemistry.

[22]  Wei He,et al.  Structural determinants for regulation of phosphodiesterase by a G protein at 2.0 Å , 2001, Nature.

[23]  D P Siderovski,et al.  Functional relevance of the disulfide-linked complex of the N-terminal PDZ domain of InaD with NorpA , 2022 .

[24]  S. Pyne,et al.  The role of G-protein coupled receptors and associated proteins in receptor tyrosine kinase signal transduction. , 2004, Seminars in cell & developmental biology.

[25]  J. Jordan,et al.  Tyrosine-kinase-dependent recruitment of RGS12 to the N-type calcium channel , 2000, Nature.

[26]  Richard R. Neubig,et al.  Regulators of G-Protein signalling as new central nervous system drug targets , 2002, Nature Reviews Drug Discovery.

[27]  Yu. Fedorov,et al.  Regulation of Myogenesis by Fibroblast Growth Factors Requires Beta-Gamma Subunits of Pertussis Toxin-Sensitive G Proteins , 1998, Molecular and Cellular Biology.

[28]  B. Snow,et al.  Molecular cloning and expression analysis of rat Rgs12 and Rgs14. , 1997, Biochemical and biophysical research communications.

[29]  D. Siderovski,et al.  RGS12 Interacts with the SNARE-binding Region of the Cav2.2 Calcium Channel* , 2005, Journal of Biological Chemistry.

[30]  Y. Peterson,et al.  Selective Interaction of AGS3 with G-proteins and the Influence of AGS3 on the Activation State of G-proteins* , 2001, The Journal of Biological Chemistry.

[31]  M. Linder,et al.  The RGS14 GoLoco Domain Discriminates among Gαi Isoforms* , 2004, Journal of Biological Chemistry.

[32]  D. Siderovski,et al.  A direct fluorescence-based assay for RGS domain GTPase accelerating activity. , 2005, Analytical biochemistry.

[33]  D. Siderovski,et al.  Purification and in vitro functional analysis of the Arabidopsis thaliana regulator of G-protein signaling-1. , 2004, Methods in enzymology.