Muscarinic receptor stimulation induces translocation of an alpha-synuclein oligomer from plasma membrane to a light vesicle fraction in cytoplasm.
The close correspondence between the distribution of brain alpha-synuclein and that of muscarinic M1 and M3 receptors suggests a role for this protein in cholinergic transmission. We thus examined the effect of muscarinic stimulation on alpha-synuclein in SH-SY5Y, a human dopaminergic cell line that expresses this protein. Under basal conditions, alpha-synuclein was detected in all subcellular compartments isolated as follows: plasma membrane, cytoplasm, nucleus, and two vesicle fractions. The lipid fractions contained only a 45-kDa alpha-synuclein oligomer, whereas the cytoplasmic and nuclear fractions contained both the oligomer and the monomer. This finding suggests alpha-synuclein exists physiologically as a lipid-bound oligomer and a soluble monomer. Muscarinic stimulation by carbachol reduced the alpha-synuclein oligomer in plasma membrane over a 30-min period, with a concomitant increase of both the oligomer and the monomer in the cytoplasmic fraction. The oligomer was associated with a light vesicle fraction in cytoplasm that contains uncoated endocytotic vesicles. The carbachol-induced alteration of alpha-synuclein was blocked by atropine. Translocation of the alpha-synuclein oligomer in response to carbachol stimulation corresponds closely with the time course of ligand-stimulated muscarinic receptor endocytosis. The data suggest that the muscarine receptor stimulated release of the alpha-synuclein oligomer from plasma membrane, and its subsequent association with the endocytotic vesicle fraction may have a role in muscarine receptor endocytosis. We propose that its function may be a transient release of membrane-bound phospholipase D2 from alpha-synuclein inhibition, thus allowing this lipase to participate in muscarinic receptor endocytosis.
Leng
Y
Y
Experimental Therapeutics Branch, NINDS, National Institutes of Health, Bethesda, Maryland 20892, USA.
Chase
T N
TN
Bennett
M C
MC
D017306:Annexin A2 / Q000378:metabolism; D015153:Blotting, Western; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D018679:Cholinergic Agonists / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism*; D004705:Endocytosis; D006801:Humans; D008049:Lipase / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D011976:Receptors, Muscarinic / Q000378:metabolism*; D051843:Synucleins; D013997:Time Factors; D051844:alpha-Synuclein
D017306:Annexin A2; D018679:Cholinergic Agonists; D009419:Nerve Tissue Proteins; D011976:Receptors, Muscarinic; C497604:SNCA protein, human; D051843:Synucleins; D051844:alpha-Synuclein; D002217:Carbachol; D008049:Lipase