A role for phosphoinositides in the regulation of muscarinic cholinergic receptor endocytosis.

An adaptive response to the prolonged agonist occupancy of many G-protein coupled receptors (GPCR), including the muscarinic cholinergic receptor (mAChR), is the endocytosis of these receptors into a vesicular compartment distinct from the plasma membrane. The purpose of this thesis was two-fold: (i) to investigate the functional status of the internalized mAChR and (ii) to determine the role that phosphoinositides play in facilitating receptor endocytosis. Incubation of SH-SY5Y neuroblastoma cells with the muscarinic agonist, Oxotremorine-M, resulted in the endocytosis of mAChRs into a vesicular (V 1) fraction. Addition of the agonist also resulted in an increased formation of the second messenger, diacylglycerol, in the V1, but this increase persisted when mAChR endocytosis was blocked. 32P phosphoinositide labeling studies and direct enzyme assays indicated a limited capacity of the V1 to synthesize phosphatidylinositol 4,5-bisphosphate (PIP 2). The latter may account for the limited ability of internalized mAChRs to activate the effector enzyme, phospholipase C (PLC). A role for phosphoinositides in the endocytosis of mAChRs was investigated via pretreatment of SH-SY5Y cells with either wortmannin (WT), LY-294002, or phenylarsine oxide (PAO). Inclusion of each of these chemically distinct inhibitors of phosphatidylinositol 4-kinase (PI4K) blocked agonist-induced mAChR endocytosis. WT-, LY-294002-, and PAO-sensitive PI4K activity was found primarily in vesicular and cytosolic fractions, consistent with the distribution of PI4K b . These results indicate that phosphoinositide synthesis regulates mAChR endocytosis and that a WT-sensitive PI4K, such as PI4K b , is required. However, overexpression of PI4K b did not effect mAChR endocytosis indicating that PI4K b is not rate-limiting. The phosphoinositide requirement for GPCR endocytosis was further investigated utilizing 1321N1 astrocytoma cells which possess PLC-coupled mAChRs and adenylyl cyclase-linked b -adrenergic receptors ( b -AR). Addition of WT alone reduced phosphatidylinositol phosphate (PIP) and PIP2 labeling and partially (30%) inhibited b -AR internalization. However, addition of Oxotremorine-M to WT-pretreated cells led to a further reduction of PIP2, but not of PIP, and to a substantial inhibition (>70%) of b -AR endocytosis. These results point to a general requirement for phosphoinositides, and PIP2 in particular, in GPCR endocytosis.PhDBiological SciencesCellular biologyHealth and Environmental SciencesNeurosciencesPharmacologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/132007/2/9938541.pd