Secretory carrier membrane proteins (SCAMPs) 1, 2, and 3: Localization, interactions, and phosphorylation

Secretory carrier membrane proteins (SCAMPs) are a family of integral membrane proteins common to all vesicular carriers comprising the cell surface recycling system. Three SCAMP isoforms, SCAMPs 1, 2, and 3 are restricted to post-Golgi compartments, where they may function together as a single protein complex.;The second series of experiments deals specifically with the tyrosine phosphorylation of a third SCAMP isoform, SCAMP3. SCAMP1 and SCAMP3 are tyrosine phosphorylated in CHO cells treated with the tyrosine phosphatase inhibitor, vanadate. Tyrosine phosphorylation can be fully reversed by vanadate washout and one hour incubation in vanadate-free media, or by incubation of tyrosine phosphorylated SCAMP3 with the recombinant tyrosine phosphatase, PTP1B. Following 5 minutes vanadate treatment, we observe a partial redistribution of SCAMP3, but not SCAMP1, from intracellular cytoplasmic vesicles to "patches" of immunostaining in the vicinity of the plasma membrane. SCAMPs 1 and 3 are tyrosine phosphorylated in epidermal growth factor (EGF)-stimulated NeoR cells, murine fibroblasts overexpressing epidermal growth factor receptor (EGFR). A timecourse of EGF stimulation reveals a low level of tyrosine phosphorylation on SCAMP 1 and SCAMP3 at 2 minutes post-stimulation, with a sustained increase in tyrosine phosphorylation up to 1 hour post-stimulation. SCAMP3 may be directly phosphorylated by EGFR in vitro. These results suggest a functional link between internalization/down-regulation of EGFR and tyrosine phosphorylation of selected SCAMPs.;Future studies need to address in more detail how SCAMPs 1, 2, and 3 function together in the regulation of vesicle trafficking and how intermolecular interactions and trafficking of SCAMPs may be regulated by one or more tyrosine kinases (e.g. EGFR) and phosphatases (e.g. PTP1B).;The first series of experiments focuses on the colocalization and interaction of SCAMPs 1 and 2. SCAMP1 and SCAMP2, extensively colocalize in membranes of fibroblasts and parotid acinar cells based on immunocytochemistry and velocity centrifugation, although the relative amounts of each variant may differ in selected organelles. SCAMP1, and to a lesser extent, SCAMP2, are substrates for chemical crosslinking in situ, and the recognizable crosslinking products of SCAMP1 suggest potential formation of homomultimers. SCAMP1 and SCAMP2 can be co-immunoprecipitated following detergent solubilization, using antibodies that specifically react with only one of the variants. Both the localization and interactions of SCAMPs are reiterated using transfected SCAMP1 that is epitope-tagged (myc) at either the NH...