Evidence that the thyrotropin receptor protease is membrane-associated and is not within lipid rafts.
The thyrotropin receptor (TSHR) cleaves to a variable extent within the ectodomain into a ligand-binding A subunit linked by disulfide bonds to the largely transmembrane B subunit. To obtain insight into this variability, we examined the extent of cleavage of TSHR ectodomains tethered to the plasma membrane by different means: (1) the wild-type, serpentine region, (2) a glycosylphosphatidylinositol (GPI) anchor, and (3) a single CD8alpha transmembrane region. For this purpose, we covalently cross-linked(125)I-TSH to the TSHR ectodomain expressed on the surface of intact cell monolayers. The extent of cleavage of the CD8alpha-tethered ectodomain was similar to the wild-type TSHR (approximately 50%) whereas the same ectodomain with a GPI anchor remained almost entirely (approximately 90%) uncleaved. These findings have three possible implications. First, differential cleavage of the TSHR ectodomain depending on its attachment to the plasma membrane suggests that the TSHR protease is membrane-associated and is not a soluble (secreted or shed) protease. Second, because GPI-anchored proteins (unlike CD8alpha) segregate in membrane lipid rafts, the TSHR protease appears not to be associated with lipid rafts. Finally, the similar extent of cleavage of the wild-type TSHR and the CD8alpha (not the GPI) tethered ectodomain supports the concept that the wild-type TSHR resides largely outside lipid rafts.
Latrofa
Francesco
F
Autoimmune Disease Unit, Cedars-Sinai Research Institute and School of Medicine, University of California, Los Angeles 90048, USA.
Chazenbalk
Gregorio D
GD
McLachlan
Sandra M
SM
Rapoport
Basil
B
D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000201:enzymology*; D006224:Cricetinae; D021962:Membrane Microdomains / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000378:metabolism*; D011989:Receptors, Thyrotropin / Q000737:chemistry / Q000378:metabolism*
D011989:Receptors, Thyrotropin; D010447:Peptide Hydrolases