A NOVEL AP180-RELATED PROTEIN IN VESICLES THAT CONCENTRATE AT ACETYLCHOLINE-RECEPTOR CLUSTERS

Monoclonal anti bodies were generated to vesicular membranes of clathr in coated vesicles enriched for acetylcholinesterase (AChE). One of th ese, C172, recognizes vesicles which accumulate in muscle cells around nuclei associated with acetylcholine receptor AChR clusters. Immunobl ots of muscle extracts and brain purified clathrin coated vesicles sho w that C172 recognizes a 100 kd band in muscle, but a 180 kd band in b rain. Western blots of purified AP180 protein stained with the two ant ibodies AP180.1 and C172 displayed the same staining pattern. Tryptic digests probed with peptide antibodies (PS26 and PS27) generated to kn own sequences of AP180 were used to map the epitope for C172 within th e brain AP180 sequence. On immunoblots of digested AP180, all AP180 an tibodies and C172 recognized a 100 kd tryptic fragment, however only C 172 recognized a smaller 60 kd. Our results suggest that the C172 epit ope is located within amino acids 305-598 of the AP180 sequence. Confo cal fluorescence microscopy of myoblasts and myotubes stained with the C172 antibody gives a punctate immunofluorescence pattern. Myoblasts stained with C172 revealed a polarized distribution of vesicles distin ct from that observed when cells are stained with gamma adaptin antibo dy which is known to localize to trans Golgi network. Myotubes stained with C172 antibody reveal a linear array of vesicular staining. Quant itative analysis of C172 reactive vesicles revealed a significant incr ease in number of vesicles present around the nuclei associated with t he acetylcholine receptor clusters. These vesicles did not colocalize with the Golgi cisternae. These results indicate that a protein with h omology to the neuron-specific coated vesicle protein AP180, is presen t in muscle cells associated with vesicles showing significant concent ration around postsynaptic nuclei present in close proximity to AChR c lusters. (C) 1998 Wiley-Liss, Inc.