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) receptor encodes a single protein with a deduced molecular mass of 84.5 kDa. This protein is cleaved during its maturation in the human thyroid since the receptor protein has been shown to be composed of two subunits (alpha subunit of approximately 53 kDa and beta subunit of approximately 38 kDa) held together by disulfide bridges [Loosfelt, H., Pichon, C., Jolivet, A., Misrahi, M., Caillou, B., Jamous, M., Vannier, B. & Miligrom, E. (1992) Proc. Natl Acad. Sci. USA 89, 3765-3769]. A similar processing occurs in an L cell line permanently expressing the human TSH receptor. The processing is however incomplete, resulting in a permanent accumulation of a 95-kDa high-mannose precursor which is present only in trace amounts in the thyroid. Pulse-chase experiments show the successive appearance in the L cells of two precursors: initially the approximately 95-kDa high-mannose glycoprotein followed by a approximately 120-kDa species containing mature oligosaccharides. This latter precursor is then processed into the alpha and beta subunits. In primary cultures of human thyrocytes precursors of similar size are detected. Spodoptera frugiperda insect cells (Sf9 and Sf21) infected with a recombinant baculovirus encoding the human TSH receptor synthesize a monomeric protein of about 90 kDa soluble only in denaturing conditions. Comparison with the product of in vitro transcription-translation experiments (approximately 80 kDa), suggests that it may be incompletely or improperly glycosylated. The TSH receptor expressed in these cells is unable to bind the hormone. Immunoelectron microscopy studies 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 surface, as well as in the endoplasmic reticulum and in the Golgi apparatus (this intracellular pool of receptor molecules probably corresponding to the high-mannose precursor); in insect cells nearly all the receptor molecules are trapped in the endoplasmic reticulum. These differences in receptor distribution are concordant with the differences observed for receptor processing.
Unité de Recherches Hormones et Reproduction, Institut National de la Santé et de la Recherche Médical, Hôpital de Bicêtre, Le Kremlin-Bicêtre, France.
M
M
Misrahi
N
N
Ghinea
S
S
Sar
B
B
Saunier
A
A
Jolivet
H
H
Loosfelt
M
M
Cerutti
G
G
Devauchelle
E
E
Milgrom
D000818:Animals; D001345:Autoradiography; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D003545:Cysteine / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure; D005057:Eukaryotic Cells / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D015151:Immunoblotting; D007739:L Cells; D008715:Methionine / Q000378:metabolism; D051379:Mice; D016253:Microscopy, Immunoelectron; D009036:Moths; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011989:Receptors, Thyrotropin / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013462:Sulfur Radioisotopes / Q000378:metabolism; D013961:Thyroid Gland / Q000378:metabolism*; D014162:Transfection
D011498:Protein Precursors; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D008715:Methionine; D003545:Cysteine