Cleavage of the thyrotropin receptor does not occur at a classical subtilisin-related proprotein convertase endoproteolytic site.
The human thyrotropin receptor (TSHR) undergoes proteolytic cleavage closely upstream to amino acid 317. Between residues 261 and 313 are three clusters of positively charged amino acids, arginines (Arg) and lysines (Lys), which are potential subtilisin-related proprotein convertase sites. We used oligonucleotide-directed mutagenesis to perform conservative amino acid substitutions within these regions (Arg or Lys to glutamine, Gln). Chinese hamster ovary cells stably transfected with mutant receptor cDNA TSHR-CS1 (Gln261) and TSHR-CS3 (Gln312, Gln313) bound radiolabeled TSH with an affinity similar to the wild-type TSHR. Mutant cDNA TSHR-CS2 (Gln290, Gln291) and TSHR-CS4 (Gln261, Gln290, Gln291, Gln312, Gln313) did not express a protein on the cell surface capable of specific TSH binding. After covalent cross-linkage of radiolabeled TSH to TSHR-CS1 and TSHR-CS3, the mutant receptors dissociated into two subunits under reducing conditions. The most prominent cluster of basic amino acids in the TSHR extracellular region (residues 287-293) was studied in a second series of mutations designed to eliminate the classical proprotein convertase sites in this region and yet be compatible with TSHR function. All three mutant receptors, TSHR-CS5 (Gln290), TSHR-CS6 (Gln291), and TSHR-CS7 (Gln291, Gln293) bound TSH with an affinity similar to that of wild type, and none of these amino acid substitutions prevented proteolytic cleavage of the extracellular domains of the TSHR. Thus, cleavage of the TSHR extracellular domain does not involve a classical subtilisin-related proprotein convertase cleavage site, raising the possibility that TSHR cleavage occurs after processing and trafficking of the protein to the plasma membrane.
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Thyroid Molecular Biology Unit, Veterans Administration Medical Center, San Francisco, California.
Rapoport
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009154:Mutation; D011989:Receptors, Thyrotropin / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*; D013381:Subtilisins / Q000378:metabolism*
D011989:Receptors, Thyrotropin; D012697:Serine Endopeptidases; D013381:Subtilisins