Regulation of m2 muscarinic receptor gene expression by platelet-derived growth factor: involvement of extracellular signal-regulated protein kinases in the down-regulation process.
To study the role of mitogen-activated protein kinase in the regulation of M2 receptors, we studied the effect of platelet-derived growth factor (PDGF) on M2 receptor gene expression. PDGF (4 ng/ml) caused a time-dependent decrease in M2 receptor number and in m2 receptor mRNA levels in HEL 299 cells. The PDGF-induced loss in m2 mRNA required de novo protein synthesis and occurred through a decrease in the rate of transcription of the m2 receptor gene. The down-regulation of M2 receptors was not accompanied by an uncoupling of the remaining receptors, indicating a large receptor reserve in these cells. Preincubations with the phosphatidylinositol 3-kinase inhibitor wortmannin, the protein kinase C inhibitor GF 109203X and the cAMP-dependent protein kinase inhibitor H-8 did not attenuate PDGF-induced down-regulation, indicating a lack of involvement of these enzymes in the down-regulation process. Activation of the extracellular signal-regulated protein kinase (ERK) 1 and 2 proteins was measured by an "in gel" phosphorylation assay. Carbachol did not activate ERK1 or 2, whereas PDGF and 4 beta-phorbol 13,14-dibutyrate resulted in a large increase in ERK1 and 2 activity along with a decrease in m2 mRNA. Preincubation with PD 098059, an inhibitor of mitogen-activated protein kinase kinase, inhibited PDGF- and 4 beta-phorbol 13,14-dibutyrate-mediated activation of ERK 1 and 2 in a concentration-dependent manner. The inhibitory action of PD 098059 was reflected at the mRNA level attenuating both PDGF- and 4 beta-phorbol 13,14-dibutyrate-mediated decreases in m2 mRNA. These results suggest a role of ERK1 and 2 in the regulation of muscarinic m2 receptor gene expression.
Rousell
J
J
Department of Thoracic Medicine, National Heart and Lung Institute, Imperial College, London, United Kingdom.
Haddad el-B
Lindsay
M A
MA
Barnes
P J
PJ
D015152:Blotting, Northern; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism / Q000502:physiology*; D000242:Cyclic AMP / Q000378:metabolism; D015536:Down-Regulation / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects* / Q000502:physiology*; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology
D010982:Platelet-Derived Growth Factor; D012333:RNA, Messenger; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic; D000242:Cyclic AMP; D011505:Protein-Tyrosine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases