Hideo Sugawa

Differences in the electrophysiological response to I− and the inhibitory anions SCN∮- and C1O4−, studied in FRTL-5 cells

The electrophysiological properties of the Na+/I- symporter (NIS) were examined in a cloned rat thyroid cell line (FRTL-5) using the whole-cell patch-clamp technique. When the holding potential was between -40 mV and -80 mV, 1 mM NaI and NaSCN induced an immediate inward current which was greater with SCN- than with I-. The reversal potential for I- and SCN- induced membrane currents was +50 mV. This is close to the value of +55 mV calculated by the Nernst equation for Na+. These results are consistent with I- and SCN- translocation via the NIS that is energized by the electrochemical gradient of Na+ and coupled to the transport of two or more Na+. There was no change in the membrane current recording with ClO-4 indicating that ClO-4 was either not transported into the cell, or the translocation was electroneutral. ClO-4 addition, however, did reverse the inward currents induced by I- or SCN-. These effects of I-, SCN- and ClO-4 on membrane currents reflect endogenous NIS activity since the responses duplicated those seen in CHO cells transfected with NIS. There were additional currents elicited by SCN- in FRTL-5 cells under certain conditions. For example at holding potentials of 0 and +30 mV, 1 mM SCN- produced an increasingly greater outward current. This outward current was transient. In addition, when SCN- was washed off the cells a transient inward current was detected. Unlike SCN-, 1-10 mM I- had no observable effect on the membrane current at holding potentials of 0 and +30 mV. The results indicate FRTL-5 cells may have a specific SCN- translocation system in addition to the SCN- translocation by the I- porter. Differences demonstrated in current response may explain some of the complicated influx and efflux properties of I-, SCN- and ClO-4 in thyroid cells.

Analysis of epidermal growth factor (EGF) receptor and effect of EGF on the growth of cultured Graves’ and non-neoplastic human thyroid cells

We analyzed epidermal growth factor receptors (EGF-R) and the growth stimulatory effects of epidermal growth factor (EGF) in the presence or absence of TSH on cultured human nonneoplastic and Graves’ thyroid cells. All cells studied possessed EGF-R composed of two components. There was no significant differences in the binding characters of EGF-R among non-neoplastic thyroid cells whether they were obtained from thyroid tissues adjacent to malignant carcinoma or benign adenoma. Ten nM of EGF stimulated (3H)-thymidine (dTR) incorporation of non-neoplastic thyroid cells by about 50%. However, TSH had no effect on the growth of these cells. Both EGF-R binding parameters and cell proliferation effects of EGF and TSH were simultaneously examined in non-neoplastic thyroid cells from 8 patients. A significant inverse correlation (r = −0.757) was observed between binding affinity (Ka1) and EGF-induced increase of dTR incorporation. Binding capacity (Cmax) did not correlate significantly with dTR incorporation. In Graves’ thyroid cells, all parameters of EGF-R were significantly lower than those of non-neoplastic thyroid cells, higher basal dTR incorporation was observed, and their goiter size significantly correlated with EGF-induced increase of dTR incorporation (r = 0.879) and also appeared to correlate inversely with Ka1. These data indicate a close relationship between the binding affinity of EGF-R and thyroid cell growth.

Aspartate-474 in the first exoplasmic loop of the thyrotropin receptor is crucial for receptor activation

Asp‐474 in the first exoplasmic loop of the thyrotropin receptor (TSHR), which is conserved among all glycoprotein hormone receptors, was mutated to Glu which is similarly charged but is longer by one methylene group and expressed in Cos‐7 cells. Cells expressing this mutant receptor showed markedly impaired TSH‐ and TSAb (thyroid stimulating antibody)‐stimulated cAMP responses with no effect on TSH binding affinity when compared with cells expressing a similar number of wild‐type receptors. These results suggest the importance of Asp‐474 in TSHR in receptor activation as demonstrated for LHR (lutropin receptor), but this, unlike LHR, is not due to the electrostatic interaction of this Asp residue with the α‐subunit Lys‐91 of the hormone.

Statistical analysis of functional region(s) of the TSH receptor

In this study, we apply recently developed methods to evaluate the thyrotropin receptor (TSH-R). These methods are called deviation (DEV) model, deviation decrease (DD) and DEV/DD analyses, and are based on deviation of amino acid sequences. A 3-dimensional structure model of TSH-R was graphically constructed, and found to possess a large central cavity (donut-like structure). The N-terminus was found to be in the center of the whole extracellular structure and to form a part of the bottom of the cavity. High DEV values indicate deviated amino acid compositions in the protein and were seen in 7 regions, 6 of which were found to be in regions with hydrophilic and acrophilic character. On the basis of the analysis of intra-molecular cis-acting relationships, 7 pairs of regions were presumed to be closely related. Further, when 3 exoplasmic loop lesions were analyzed similarly, 3 other regions were shown to have a close relationship with the cell surface. DEV/DD values were applied to predict the interface of TSH-R with trans-acting molecules such as TSH-R antibody or TSH. The regions in association with trans-acting molecules were seen in 14 regions, 11 of which included the high DEV regions. Both of the TSH-R specific regions in the N- and C-terminal side, especially the latter, were found to be the major components.