PROCESSING OF THE PRECURSORS OF THE HUMAN THYROID-STIMULATING HORMONE-RECEPTOR IN VARIOUS EUKARYOTIC CELLS (HUMAN THYROCYTES, TRANSFECTED L-CELLS AND BACULOVIRUS-INFECTED INSECT CELLS)

The complementary DNA for human thyroid-stimulating hormone (TSH) rece ptor encodes a single protein with a deduced molecular mass of 84.5 kD a. This protein is cleaved during its maturation in the human thyroid since the receptor protein has been shown to be composed of two subuni ts (alpha subunit of approximate to 53 kDa and beta subunit of approxi mate to 38 kDa) held together by disulfide bridges [Loosfelt, H., Pich on, C., Jolivet, A., Misrahi, M., Caillou, B., Jamous, M., Vannier, B. and Milgrom, E. (1992) Proc. Natl Acnd. Sci. USA 89, 3765-3769]. A si milar processing occurs in an L cell line permanently expressing the h uman TSH receptor. The processing is however incomplete, resulting in a permanent accumulation of a 95-kDa high-mannose precursor which is p resent only in trace amounts in the thyroid. Pulse-chase experiments s how the successive appearance in the L cells of two precursors: initia lly the approximate to 95-kDa high-mannose glycoprotein followed by a approximate to 120-kDa species containing mature oligosaccharides. Thi s latter precursor is then processed into the alpha and beta subunits. In primary cultures of human thyrocytes precursors of similar size ar e detected. Spodoptera frugiperda insect cells (Sf9 and Sf21) infected with a recombinant baculovirus encoding the human TSH receptor synthe size a monomeric protein of about 90 kDa soluble only in denaturing co nditions. Comparison with the product of in vitro transcription-transl ation experiments (approximate to 80 kDa), suggests that it may be inc ompletely or improperly glycosylated. The TSH receptor expressed in th ese cells is unable to bind the hormone. Immunoelectron microscopy stu dies show that in human thyrocytes most of the receptor is present on the cell surface; in L cells the receptor is detected on the cell surf ace, as well as in the endoplasmic reticulum and in the Golgi apparatu s (this intracellular pool of receptor molecules probably correspondin g to the high-mannose precursor); in insect cells nearly all the recep tor molecules are trapped in the endoplasmic reticulum. These differen ces in receptor distribution are concordant with the differences obser ved for receptor processing.