Role of amino acids 261-418 in proteolytic cleavage of the extracellular region of the human thyrotropin receptor.
Previous in vivo cross-linking studies of TSH to the recombinant TSH receptor revealed that the receptor exists at least in part as a single chain glycoprotein of approximately about 100 kilodaltons (kDa), with intramolecular disulfide bonds. TSH also binds to a 54-kDa amino-terminal fragment of the TSH receptor (cleaved up-stream of amino acid residue 317). In the present study in order to better understand the structure of the TSH receptor, we covalently cross-linked radiolabeled TSH to six TSH-LH receptor extracellular region chimeras and the wild-type TSH receptor expressed on Chinese hamster ovary cells in vivo. In these chimeras, different regions of the TSH receptor were substituted with the homologous regions of the LH receptor. When analyzed under nonreducing conditions by polyacrylamide gel electrophoresis, the TSH-cross-linked products were similar to all TSH-LH receptor chimeras and the wild-type receptor. In contrast, differences among the receptors were noted when the TSH-cross-linked products were examined under reducing conditions. With the exception of two chimeras, as noted previously with the wild-type receptor, two TSH-cross-linked products were observed, representing TSH cross-linked to a holoreceptor of about approximately 100 kDa and a fragment of the receptor of about approximately 54 kDa. However, in the two chimeras in which both domains D and E (amino acids 261-418) of the TSH receptor were substituted, only the holoreceptor and not the smaller fragment was detected. Substitution of domains ABC (amino acids 1-260) did not prevent proteolytic cleavage of the TSH receptor. In conclusion, amino acids 261-418 are necessary for proteolytic cleavage of the extracellular region of the human TSH receptor.
Russo
D
D
Thyroid Molecular Biology Unit, Veterans Administration Medical Center, San Francisco, California.
Nagayama
Y
Y
Chazenbalk
G D
GD
Wadsworth
H L
HL
Rapoport
B
B
D010450:Endopeptidases / Q000502:physiology; D006801:Humans; D011974:Receptors, LH / Q000737:chemistry / Q000378:metabolism; D011989:Receptors, Thyrotropin / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship
D011974:Receptors, LH; D011989:Receptors, Thyrotropin; D010450:Endopeptidases