Identification and Evaluation of Constitutively Active Thyroid Stimulating Hormone Receptor Mutations

Thyroid stimulating hormone receptor (TSHR) is a guanine nucleotide-binding protein-coupled seven-transmembrane-domain receptor that controls the differentiation, growth, and function of the thyroid gland through stimulation of adenylyl cyclase and phospholipase C pathways. Thyroid stimulating hormone (TSH) is the main TSHR ligand, and unliganded receptor remains silent due to the interaction of its large extracellular domain with the extracellular loops of the serpentine. The TSHR gene is highly mutagenic and constitutively active mutations have been extensively described. Naturally occurring TSHR-activating mutations can affect any part of the receptor, but most activating mutations affect the serpentine region, and the majority of these are located in the third intracellular loop or transmembrane domain six. We describe several simple and relatively cheap methods used in our laboratory to study constitutive TSHR mutations that include (1) screening of TSHR gene mutations in paraffin-embedded thyroid tissue samples, (2) measurement of TSHR constitutive activity in vitro, (3) measurement of TSHR expression at cell surface by flow cytometry analysis, (4) TSH binding to TSHR, and (5) TSHR phosphorylation analysis .