Subunit Interactions Influence TSHR Multimerization

The TSH receptor (TSHR) is the key molecule influencing thyroid growth and development and is an antigenic target in autoimmune thyroid disease. The TSHR exists in monomeric and multimeric forms, and it has been shown previously that multimeric complexes of the TSHR preferentially localize in lipid rafts. However, unlike other glycoprotein hormone receptors, the TSHR exists in several forms on the cell membrane due to intramolecular cleavage of its ectodomain, which causes the production of α- and β-subunits of various lengths. After cleavage and reduction of disulfide bonds, α-subunits consisting of the receptor ectodomain may be lost from the cell surface by receptor shedding, leading to accumulation of excess β-subunits within the membrane. Because cell surface expression of these various forms of the TSHR is critical to receptor signaling and autoimmune responses, we set out to model the influence of β-subunits on full-length TSHRs. To study this interaction, we generated three truncated ectodomain β-subunits linked to green fluorescent protein (named β-316, -366, and -409) as examples of native cleaved forms of the TSHR. These constructs were transfected into human embryonic kidney 293 cells in the presence and absence of the full-length receptor. Whereas the β-316 and β-366 forms showed cell surface expression, the expression of β-409 was primarily intracellular. Cotransfection of the β-subunits with a full-length hemagglutinin-tagged wild-type (WT) receptor (HT-WT-TSHR) in both transient and stable systems caused a significant decrease in surface expression of the full-length WT receptors. This decrease was not seen with control plasmid consisting of a plasma membrane-targeted protein tagged to red fluorescent protein. To ascertain if this response was due to homointeraction of the truncated β-constructs with the WT-TSHRs, we immunoprecipitated membranes prepared from the cotransfected cells using antihemagglutinin and then probed with anti-green fluorescent protein. These studies confirmed dimerization of the β-subunits with the WT full-length receptor, and this interaction was further observed in vivo by fluorescence resonance energy transfer. We then studied the functional consequences of this interaction on TSHR signaling by examining Gαs-mediated signals. The well-expressed truncated constructs, when coexpressed with full-length TSHR, did not alter constitutive cAMP levels, but there was a significant decrease in TSH-induced cAMP generation. Furthermore, we observed that truncated β-316 and β-366 had faster internalization rate, which may lead to a significant decrease in the expression of the full-length receptor on the cell surface, thus contributing to the decreased signaling response. However, the decrease in surface receptors may also be due to inhibition of newly formed receptors reaching the surface as result of receptor-receptor interaction. It is well known that under normal physiological conditions both cleaved and uncleaved TSHR forms coexist on the cell surface of normal thyrocytes. Our studies allow us to conclude, therefore, that multimerization of cleaved/ truncated forms of the β-subunits with the full-length TSHR has a profound influence on TSHR internalization and signaling. Hence, the degree of intramolecular cleavage must also modulate TSHR signaling.