p145, a major Grb2-binding protein in brain, is co-localized with dynamin in nerve terminals where it undergoes activity-dependent dephosphorylation.
Three major rat brain proteins were recently found to bind the SH3 domains of Grb2: synapsin I, dynamin, and a novel 145-kDa protein (p145) (McPherson, P. S., Czernik, A. J., Chilcote, T. J., Onofri, F., Benfenati, F., Greengard, P., Schlessinger, J., and De Camilli, P. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 6486-6490). We have now used antibodies raised against p145 which had been purified by Grb2 affinity chromatography and SDS-polyacrylamide gel electrophoresis to characterize this protein. p145 is neuron-specific and co-distributes with dynamin in subcellular fractions of rat brain. Both proteins are partially recovered in soluble and particulate fractions. By immunofluorescence, p145 is closely co-localized with dynamin, which we show here to be highly concentrated in nerve terminals. In contrast to synapsin I, which is highly enriched in a purified synaptic vesicle fraction, both p145 and dynamin are de-enriched in this fraction. However, both proteins are found on membranes which are immunoisolated with antibodies directed against the synaptic vesicle membrane protein synaptophysin, suggesting that dynamin and p145 are localized in part on organelles which represent intermediate stages in the reformation of synapatic vesicles during recycling. Finally, we have determined that p145, like dynamin, is a phosphoprotein which undergoes dephosphorylation in response to nerve terminal depolarization. These findings suggest that p145 participates with dynamin in synaptic vesicle endocytosis and recycling.
McPherson
P S
PS
Department of Cell Biology, Howard Hughes Medical Research Institute, Yale University School of Medicine, New Haven, Connecticut 06510.
Takei
K
K
Schmid
S L
SL
De Camilli
P
P
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001921:Brain / Q000378:metabolism* / Q000648:ultrastructure; D001923:Brain Chemistry; D002458:Cell Fractionation; D034281:Dynamins; D004591:Electrophoresis, Polyacrylamide Gel; D066246:ErbB Receptors / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D051380:GRB2 Adaptor Protein; D020558:GTP Phosphohydrolases / Q000302:isolation & purification / Q000378:metabolism*; D008297:Male; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism; D008970:Molecular Weight; D009411:Nerve Endings / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D009457:Neuroglia / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D009928:Organ Specificity; D015388:Organelles / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D013574:Synaptosomes / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495950:Grb2 protein, rat; D011506:Proteins; D066246:ErbB Receptors; D020558:GTP Phosphohydrolases; D034281:Dynamins