Involvement of TSHR-hinge region and hormone α-subunit in hormone mediated conformational change in TSHR.

<p>Flow cytometric analysis of binding of 25 µg/ml of (<b>9A.</b>) MAb 311.62 or (<b>9B.</b>) MAb 311.82 <i>to</i> HEK293-hTSHR cells in absence of TSH (shaded solid line histogram) or pre-incubated with either 1 nM TSH (shaded dotted line histogram) or 25 nM TSH (shaded dashed line histogram). The culture supernatant of hormone α-subunit specific monoclonal antibody 2A6 was either added to HEK293-hTSHR cells previously incubated with (<b>9C.</b>) 25 nM TSH or (<b>9D.</b>) 25 nM TSH was added to HEK293-hTSHR cells previously exposed to similar concentration of 2A6 and binding of 2A6 to TSH in a TSH-TSHR complex was monitored in flow cytometry. All the flow cytometric experiments were carried out at 4°C to prevent hormone mediated receptor internalization.