This application seeks to convert a NIRA (R23) Award to a FIRST Award (R29). The objective of the proposed research is to determine the subcellular locations of the post-translational processing of thyroid-stimulating hormone (TSH) subunit precursors. Mouse thyrotropic tumor will be utilized as a source of actively secreting thyrotrophs; pituitary tissue of euthyroid and thyroidectomized mice will be sources of """"""""resting"""""""" and """"""""stimulated"""""""" thyrotrophs, respectively. Tumor and pituitary minces will be incubated in vitro with (3H)-or (35S) methionine, (3H) fucose, (35S) sulfate, (3H) N-acetylmannosamine, or (14C)-or (3H) mannose, and then homogenized and fractionated by the sucrose step-gradient method that we have used for previous studies. Cell fractions enriched in rough endoplasmic reticulum and Golgi will be characterized by electron microscopy. Carboxyl cyanide m-chlorophenylhydrazone (CCCP) and monensin will be used as adjuncts to subcellular fractionation to make inferences about the subcellular location of processing events. Labeled TSH subunits will be immunoprecipitated from cell homogenates, cell fractions, and media, and will be characterized by SDS- polyacrylamide gel electrophoresis. (3H) mannose-labeled oligosaccharides will be released from TSH subunits by endoglycosidase H, and their structures determined by paper chromatography. Dual-labeled tryptic glycopeptides will be characterized by HPLC. We will learn whether the three tissue types differ in the subcellular locations or nature of processing events. We will determine for each compartment of each tissue type the extent and kinetics of alpha-beta subunit combination, the molar ratios of subunits, and the subunit content of fucose, sialic acid, and sulfate; glycopeptide analyses will determine the degree of fucosylation, sialylation, or sulfation of the complex oligosaccharides, or the high mannose structures, at each asparaginyl glycosylation site of TSH. Unlike studies of glycoprotein processing by others, we propose to study a hormonally-responsive system in which the oligosaccharides of a major secreted species, TSH, are believed to be important for biological activity. Our findings will be clinically relevant to rare patients with hypothalamic hypothyroidism and pituitary TSH- producing tumors. Primarily, we hope to contribute to a better understanding of the basic cell biology of TSH biosynthesis.
