Agonist-induced endocytosis of muscarinic cholinergic receptors: relationship to stimulated phosphoinositide turnover.
The ability of muscarinic cholinergic receptors to activate phosphoinositide turnover following agonist-induced internalization has been investigated. Incubation of SH-SY5Y neuroblastoma cells with oxotremorine-M resulted in a time-dependent endocytosis of both muscarinic receptors and alpha subunits of G0 and G11, but not of isoforms of phosphoinositide-specific phospholipase C, into a subfraction of smooth endoplasmic reticulum (V1). Agonist-induced increases in diacylglycerol mass and in 32P-phosphatidate labeling, much of which was of the tetraenoic species, were also observed in the V1 fraction, but these increases persisted when the agonist-induced translocation of receptors into the V1 fraction was blocked. All enzymes of the phosphoinositide cycle were detectable in the V1 fraction. However, with the exception of phosphatidylinositol 4-kinase, none was enriched when compared with cell lysates. Both 32P-labeling studies and enzyme assays point to a very limited capacity of this fraction to synthesize phosphatidylinositol 4,5-bisphosphate, whereas the synthesis of phosphatidylinositol 4-phosphate is robust. These results indicate that endocytosed receptors do not appear to retain their ability to activate phosphoinositide turnover. The availability of the substrate for phospholipase C, phosphatidylinositol 4,5-bisphosphate, may be one factor that limits the activity of muscarinic receptors in this subcellular compartment.
Sorensen
S D
SD
Neuroscience Laboratory, University of Michigan, Ann Arbor 48104-1687, USA.
McEwen
E L
EL
Linseman
D A
DA
Fisher
S K
SK
D015153:Blotting, Western; D004075:Diglycerides / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects*; D005910:Glioma; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D050356:Lipid Metabolism; D018721:Muscarinic Agonists / Q000494:pharmacology*; D009447:Neuroblastoma; D010095:Oxotremorine / Q000031:analogs & derivatives / Q000494:pharmacology; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000032:analysis / Q000378:metabolism*; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000166:cytology / Q000201:enzymology; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism
C011439:1,2-diacylglycerol; D004075:Diglycerides; D007527:Isoenzymes; D018721:Muscarinic Agonists; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D011976:Receptors, Muscarinic; D010095:Oxotremorine; C042743:oxotremorine M; D010738:Type C Phospholipases