THYROTROPIN STIMULATION OF THE LUTROPIN CHORIOGONADOTROPIN RECEPTOR -DIFFERENT SITES MEDIATE AGONIST ACTIVITY AND HIGH-AFFINITY BINDING/

Surprisingly, thyrotropin (TSH) can increase cAMP and inositol phospha te (IP) levels in Cos-7 cells transfected with the hutropin (LH)/chori ogonadotropin (CG) receptor (LH/CGR) as well as LH or CG, as evidenced by similar EC(50) and maximal stimulation values. Additionally surpri sing, TSH activation is evident, despite markedly reduced levels of hi gh affinity TSH binding by comparison to CG (Hidaka A, et al. 1993 Bio chem Biophys Res Commun 196:187-195). In this report, we questioned wh ether the unusual TSH activity, as well as the discrepancy between TSH activity and binding, might reflect the existence of distinct agonist and binding sites on the LH/CGR extracellular domain and the ability of TSH to interact with the former despite a minimal interaction with the latter. We evaluated this possibility by using two chimeras spanni ng the extracellular domain of the TSHR and the LH/CGR: Mc1 + 2, where residues 8-165 of the TSHR are substituted, and Mc2 + 3 + 4, where re sidues 90-370 are replaced with the corresponding peptide segment from the LH/CGR. After transfection in Cos-7 cells, Mc2 + 3 + 4 exhibits h igher affinity for CG than wild-type LH/CGR, but has no CG agonist res ponse in assays measuring cAMP or inositol phosphate (IP) levels. Conv ersely, the Mc1 + 2 chimera exhibits significantly decreased affinity for CG, but CG agonist activity Is comparable to wild-type LH/CGR in c AMP and IP assays. These data show that the extracellular domain of th e LH/CGR does have distinct sites for CG binding and agonist activity: the C-terminus in Mc2 + 3 + 4 is important for high affinity CG bindi ng, whereas the N-terminus in Mc1 + 2 is able to exhibit a CG agonist response, despite low affinity binding. When evaluated using TSH, Mc1 + 2,with the C-terminus of the TSHR present, exhibits high affinity TS H binding comparable to wild-type TSHR. Unexpectedly, Mc1 + 2, with th e substitution of the N-terminus of the extracellular domain of the LH /CGR, exhibits even better TSH agonist activity than wild-type TSHR, n ot a loss of activity. Thus, the N-terminus of the extracellular domai n of the LH/CGR can couple TSH binding to signal transduction events e ven better than the N-terminus of the TSHR. This may, in part, explain why TSH has an unusual agonist activity in cells transfected with LH/ CGR, despite relatively low affinity binding. Although distinct agonis t and binding sites exist in the linear sequence of the extracellular domain, the activity of the two sites is interdependent. Thus, Mc1 + 2 retains the linear epitopes for reactivity with blocking TSHR autoant ibodies; however, the ability of these antibodies to inhibit TSH-incre ased cAMP levels is reduced by comparison to wild-type TSHR. Three of the four blocking TSHR IgGs were, in addition able to inhibit hCG incr eased cAMP levels in Mc1 + 2-transfected cells better than inhibition of TSH-stimulated cAMP levels in the same cells. There is, therefore, an altered interaction between the N- and C-terminus in Mc1 + 2; as a result, high affinity TSH binding overcomes the blocking TSHRAb inhibi tion, whereas low affinity hCG binding does not and inhibition persist s.