SEROTONIN BINDING-PROTEIN - SYNTHESIS, SECRETION, AND RECYCLING

Serotonin binding protein (SBP) is present in all neurectodermally der ived cells that store serotonin (5-HT). Three forms of SBP have been d etected (68, 56, and 45 kDa), and antibodies to SBP that interfere wit h the binding of 5-HT react with each of these proteins. The current e xperiments test two hypotheses: (a) that the 56- and 45-kDa forms of S BP are produced by posttranslational cleavage of a 68-kDa precursor mo lecule; and (b) that 45-kDa SBP is a constituent of serotonergic secre tory vesicles. Pulse-chase experiments were carried out using medullar y thyroid carcinoma cells as a model. These neurectodermally derived c ells produce 5-HT and all three forms of SBP. Following pulse labeling for 20 min with L-[S-35]methionine, the cells were incubated in the p resence of an excess of unlabeled L-methionine for 0, 30, 60, or 90 mi n at 37 degrees C. Alternatively, the chase was performed under condit ions (20 degrees C, inhibition of ATP generation) that delay or stop t ransport of newly synthesized proteins from the rough endoplasmic reti culum through the Golgi apparatus. Following incubation, the cells wer e washed and solubilized, and SBP was immunoprecipitated. Radioactive proteins in the immunoprecipitate were electrophoretically resolved an d quantified. Immediately after the pulse, each of the three forms of SBP was found to be labeled with S-35. The relative proportions of S-3 5-labeled 68-, 56-, and 45-kDa SBP remained the same at each interval of chase. These proportions were not changed when the chase was carrie d out at 20 degrees C or under conditions that blocked the biosynthesi s of ATP. These observations suggest that each form of SBP is a primar y product of translation, that the smaller forms of SBP are not produc ed by cleavage from a larger molecule, and that the size of the primar y products of translation is not altered by passage to the Golgi appar atus or a post-Golgi compartment. When secretion was induced, 45-kDa S BP, but not 56- or 68-kDa SBP, was released to the medium. When antibo dies to 45-kDa SBP were added to the medium at the time secretion was induced, antibody binding sites appeared as patches on the cell surfac es. Because of these sites, cells were lysed when they were stimulated to secrete in the presence of antibodies to 45-kDa SBP and guinea pig complement. Antibody binding sites disappeared from cell surfaces aft er 20 min, at which time antibodies to SBP were found inside the cells . It is suggested that 45-kDa SBP is packaged with 5-HT in secretory v esicles. Some 45-kDa SBP is lost during secretion as a result of exocy tosis; however, a fraction of the 45-kDa SBP remains bound to the lumi nal surface of the membrane of secretory vesicles. This protein is exp osed to the ambient medium as a consequence of exocytosis, but is rein ternalized when the vesicular membrane is recaptured during vesicle re cycling.