Linkage of the acetylcholine transporter-vesamicol receptor to proteoglycan in synaptic vesicles.
The relationship of the acetylcholine transporter-vesamicol receptor (AcChT-VR) to proteoglycan in Torpedo electric organ synaptic vesicles was investigated. The cholate-solubilized VR was immunoprecipitated by a monoclonal antibody directed against the SV1 epitope located in the glycosaminoglycan portion of the proteoglycan. AcChT that was photoaffinity-labeled with a tritiated high-affinity analogue of AcCh [cyclohexylmethyl cis-N-(4-azidophenacyl)-N-methylisonipecotate] and then denatured in sodium dodecyl sulfate also immunoprecipitated. The labeled AcChT exhibited a M(r) range of 100,000-200,000. Proteoglycan did not engage in detectable nonspecific reversible aggregation that might mask the presence of another subunit during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In vesicles permeabilized with cholate, the enzymes keratanase and testicular hyaluronidase inactivated binding of vesamicol and destroyed the SV1 epitope without detectable proteolysis. Other glycosaminoglycan-degrading enzymes were without effect. The results demonstrate that the AcChT-VR and proteoglycan are very strongly linked and that glycosaminoglycan-like polysaccharide controls the conformation of the VR. The unexpected linkage to proteoglycan suggests that AcChT-VR in intact terminals might communicate with extracellular matrix and participate in stabilization and operation of the synapse.
Bahr
B A
BA
Department of Chemistry, University of California, Santa Barbara 93106.
Noremberg
K
K
Rogers
G A
GA
Hicks
B W
BW
Parsons
S M
SM
D000109:Acetylcholine / Q000031:analogs & derivatives / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000378:metabolism; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D004557:Electric Organ / Q000378:metabolism* / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases; D006821:Hyaluronoglucosaminidase / Q000494:pharmacology; D007163:Immunosorbent Techniques; D008297:Male; D026901:Membrane Transport Proteins; D010777:Photochemistry; D011509:Proteoglycans / Q000378:metabolism*; D017981:Receptors, Neurotransmitter / Q000187:drug effects / Q000378:metabolism*; D017478:Receptors, Phencyclidine; D013572:Synaptic Vesicles / Q000378:metabolism*; D013737:Testis / Q000201:enzymology; D014101:Torpedo; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins; D001616:beta-Galactosidase / Q000494:pharmacology
D000345:Affinity Labels; D001386:Azides; D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D011509:Proteoglycans; D017981:Receptors, Neurotransmitter; D017478:Receptors, Phencyclidine; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins; C075303:cyclohexylmethyl-N-(4-azidophenacyl)-N-methylisonipecotate; D006026:Glycoside Hydrolases; C026093:keratan-sulfate endo-1,4-beta-galactosidase; D001616:beta-Galactosidase; D006821:Hyaluronoglucosaminidase; D000109:Acetylcholine