title: An N-linked glycosylation motif from the noncleaving luteinizing hormone receptor substituted for the homologous region (Gly367 to Glu369) of the thyrotropin receptor prevents cleavage at its second, downstream site.
abstract: The thyrotropin receptor (TSHR) exists in two forms (single polypeptide and two subunits), whereas the lutropin/chorionic gonadotropin receptor (LH/CGR) is a single chain. Recent data suggest that the TSHR cleaves at two sites. We mutagenized selected chimeric TSH-LH/CGR to localize the cleavage sites in the TSHR. All 23 receptors mutated in the estimated vicinity of the upstream site cleaved into two subunits as determined by 125I-TSH cross-linking to intact cells. In contrast, in a series of mutations homologous to the noncleaving LH/CGR, the downstream TSHR cleavage site localized to three amino acids (GQE367-369). Remarkably, group substitution of these residues, but not substitution of individual residues, abolished cleavage. Moreover, the mutation that prevented cleavage (GQE367-369NET) transposed a motif (NET291-293) that is glycosylated in the LH/CGR. TSHR cleavage or noncleavage after substitution of GQE367-369 with other triplets (AAA, NQE, and NQT) was consistent with a role for N-linked glycosylation at this site. In summary, our data (i) support the concept that the TSHR cleaves at two sites, (ii) relate TSHR residues GQE367-369 to cleavage at the second, downstream site, and (iii) suggest that cleavage or noncleavage at site two is related to N-linked glycosylation. These findings provide new insight into the evolutionary divergence of two closely related receptors.
authors.affiliation: Thyroid Molecular Biology Unit, Veterans Administration Medical Center and the University of California, San Francisco, California 94121, USA.
authors.forename: A
authors.initials: A
authors.lastname: Kakinuma
authors.forename: G D
authors.initials: GD
authors.lastname: Chazenbalk
authors.forename: K
authors.initials: K
authors.lastname: Tanaka
authors.forename: Y
authors.initials: Y
authors.lastname: Nagayama
authors.forename: S M
authors.initials: SM
authors.lastname: McLachlan
authors.forename: B
authors.initials: B
authors.lastname: Rapoport
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002417:Cattle; D005973:Glutamine / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D006031:Glycosylation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011974:Receptors, LH / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011989:Receptors, Thyrotropin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D011974:Receptors, LH; D011989:Receptors, Thyrotropin; D011993:Recombinant Fusion Proteins; D005973:Glutamine; D005998:Glycine