AGONIST-INDUCED ENDOCYTOSIS OF MUSCARINIC CHOLINERGIC RECEPTORS - RELATIONSHIP TO STIMULATED PHOSPHOINOSITIDE TURNOVER

The ability of muscarinic cholinergic receptors to activate phosphoino sitide turnover following agonist-induced internalization has been inv estigated. Incubation of SH-SY5Y neuroblastoma cells with oxotremorine -M resulted in a time-dependent endocytosis of both muscarinic recepto rs and alpha subunits of G(q) and G(11), but not of isoforms of phosph oinositide-specific phospholipase C, into a subfraction of smooth endo plasmic reticulum (V-1). Agonist-induced increases in diacylglycerol m ass and in P-32-phosphatidate labeling, much of which was of the tetra enoic species, were also observed in the V-1 fraction, but these incre ases persisted when the agonist-induced translocation of receptors int o the V-1 fraction was blocked. All enzymes of the phosphoinositide cy cle were detectable in the V-1 fraction. However, with the exception o f phosphatidylinositol 4-kinase, none was enriched when compared with cell lysates. Both P-32-labeling studies and enzyme assays point to a very limited capacity of this fraction to synthesize phosphatidylinosi tol 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 tu rnover. The availability of the substrate for phospholipase C, phospha tidylinositol 4,5-bisphosphate, may be one factor that limits the acti vity of muscarinic receptors in this subcellular compartment.