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To gain insight into the thyrotropin hormone (TSH) receptor (TSHR) cleavage, we sought to convert the noncleaving luteinizing hormone (LH) receptor (LHR) into a cleaved, two-subunit molecule. For this purpose, we generated a series of LHR mutants and chimeric LH-TSH receptors. Cleavage of mature, ligand binding receptors on the cell surface was determined by covalent 125I-labeled hCG crosslinking to intact, stably transfected mammalian cells. We first targeted a cluster of three N-linked glycans in the LHR (N295, N303, N317) in a region corresponding to the primary TSHR cleavage site, which has only one N-linked glycan. Elimination by mutagenesis of the most strategic N-linked glycan (LHR-N317Q) generated only a trace amount of LHR cleavage. Removal of the other N-linked glycans had no additive effect. A much greater degree of cleavage ( approximately 50%) was evident in a chimeric LH-TSHR in which the juxtamembrane segment of the LHR (domain E; amino acids 317-367) was replaced with the corresponding domain of the TSHR (residues 363-418). Similarly cleaving LHR were created using a much smaller component within this region, namely LHR-NET317-319 replaced with TSHR-GQE367-369, or by substitution of the same three amino-acid residues with AAA (LHR-NET317-319AAA). In summary, our data alter current concepts regarding TSHR cleavage by suggesting limited (not absent) amino-acid specificity in a region important for TSHR cleavage (GQE367-369). The data also support the concept of a separate and distinct downstream cleavage site 2 in the TSHR.

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.

We examined the relative effects of thyrotropin (TSH) and TSH receptor autoantibodies in the sera of patients with autoimmune thyroid disease on three TSH-lutropin/chorionic gonadotropin (LH/CG) receptor extracellular domain chimeras. Each chimera binds TSH with high affinity. Only the chimera with TSH receptor extracellular domains ABC (amino acids 1-260) had a functional (cAMP) response to thyroid stimulatory IgG. The chimeras with TSH receptor domains CD (amino acids 171-360) and DE (amino acids 261-418) were unresponsive. The lack of response of the chimera with TSH receptor domains DE was anticipated because it fails to transduce a signal with TSH stimulation, unlike the other two chimeras. A different spectrum of responses occurred when the TSH-LH/CG chimeras were examined in terms of autoantibody competition for TSH binding. IgG with TSH binding-inhibitory activity when tested with the wild-type TSH receptor also inhibited TSH binding to the chimera with TSH receptor domains DE. Dramatically, however, these IgG did not inhibit TSH binding to the chimera with TSH receptor domains CD, and had weak or absent activity with the chimera with TSH receptor domains ABC. Chimeras with TSH receptor domains ABC and DE were equally effective in affinity-purifying IgG with thyroid-stimulatory and TSH binding-inhibitory activities. Nonstimulatory IgG with TSH binding-inhibitory activity inhibited the action of stimulatory IgG on the wild-type TSH receptor, but not with the chimera containing TSH receptor domains ABC. In summary, TSH receptor autoantibodies and TSH bind to regions in both domains ABC and DE of the TSH receptor extracellular region. Stimulatory and inhibitory TSH receptor autoantibodies, as well as TSH, appear to bind to different sites in domains ABC, but similar sites in domains DE, of the receptor. Alternatively, TSH and the different TSH receptor antibodies bind with differing affinities to the same site in the ABC region.

To define the sites in the extracellular domain of the human thyrotropin (TSH) receptor that are involved in TSH binding and signal transduction we constructed chimeric thyrotropin-luteinizing hormone/chorionic gonadotropin (TSH-LH/CG) receptors. The extracellular domain of the human TSH receptor was divided into five regions that were replaced, either singly or in various combinations, with homologous regions of the rat LH/CG receptor. The chimeric receptors were stably expressed in Chinese hamster ovary cells. The data obtained suggest that the carboxyl region of the extracellular domain (amino acid residues 261-418) and particularly the middle region (residues 171-260) play a role in signal transduction. The possibility is also raised of an interaction between the amino and carboxyl regions of the extracellular domain in the process of signal transduction. With respect to hormone binding, substitution of the entire extracellular domain of the LH/CG receptor for the corresponding region of the TSH receptor resulted in high-affinity human CG binding with complete loss of TSH binding. Surprisingly, however, there was at least one chimera with a substitution at each of the five domains that still retained high-affinity TSH binding. Substitution of residues 1-170 of the TSH receptor with the corresponding region of the LH/CG receptor was associated with the retention of high-affinity TSH binding but ligand specificity was lost in that TSH and human CG could interact functionally with the receptor. In summary, these studies suggest that the middle region and carboxyl half of the extracellular domain of the TSH receptor are involved in signal transduction and that the TSH-binding region is likely to span the entire extracellular domain, with multiple discontinuous contact sites.

We constructed a series of TSH-LH/CG receptor chimeras by homologous substitution of relatively small regions of the TSH receptor extracellular domain for the corresponding region of the extracellular domain of the LH/CG receptor. Constructs were stably expressed in Chinese hamster ovary cells. Of the five chimeric receptors, only TSH-LHR-14, which contains mid-region domain C (amino acid residues 171-260) of the extracellular component of the TSH receptor, exhibited TSH binding of relatively high affinity. Consistent with this TSH binding, chimera TSH-LHR-14 was the only one that demonstrated a functional response to TSH stimulation in terms of intracellular cAMP generation. These data indicate that domain C plays a vital role in TSH receptor function.