Immunoprecipitation isolates multiple TSH receptor forms from human thyroid tissue.
Attempts to purify the thyroid-stimulating hormone receptor (TSHR) have been complicated by its susceptibility to proteolysis and its low level of expression in thyrocytes and many transfected cells. Controversy exists over its size and structure. Multiple, single-polypeptide forms of the TSHR (230, 187, and 95-100 kDa) have been recently identified in immunoblots of crude plasma membranes prepared from COS-7 cells transfected with rat or human cDNA, but the relationship of these receptor species to the TSHR in human thyroid tissue has been heretofore unknown. We have developed a technique for immunoprecipitation of the TSHR which employed IgG purified from a polyclonal rabbit antiserum to TSHR residues 352-366. We have used immunoprecipitation to isolate the previously characterized 95-100 kDa TSH-holoreceptor, 187 kDa intermediate, and 230 kDa precursor forms of the TSHR from plasma membrane prepared from transfected COS-7 cells and human thyroid tissue. The presence of all three forms was not altered by the addition of reducing agent to the sample buffer, demonstrating the single polypeptide structure of the TSHR. This is, to our knowledge, the first report of the purification from transfected COS-7 cells of these TSHR species identified previously only in immunoblots of crude plasma membrane, and the first report of the identification by any means of these TSHR forms in human thyroid tissue. The isolation of TSHR from human thyroid tissue requires confirmation by direct means, but promises to make the receptor available in a soluble form for studies of its structure and function.
Grossman
R F
RF
Dept. of Surgery, Mount Zion Medical Center of UCSF, USA.
Ban
T
T
Duh
Q Y
QY
Tezelman
S
S
Jossart
G
G
Soh
E Y
EY
Clark
O H
OH
Siperstein
A E
AE
D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D007536:Isomerism; D008970:Molecular Weight; D011233:Precipitin Tests; D011989:Receptors, Thyrotropin / Q000302:isolation & purification*; D013961:Thyroid Gland / Q000737:chemistry*; D014162:Transfection
D011989:Receptors, Thyrotropin