A Fast Method to Detect Cell Surface Expression of Thyrotropin Receptor (TSHr): The Microchip Flow Cytometry Analysis Patrizia Agretti, 1 Giuseppina De Marco, 1 Alessandra Capodanno, 2 Eleonora Ferrarini, 1 Antonio Dimida, 1 Daniela Sansone, 1 Paola Collecchi, 2 Aldo Pinchera, 1 Paolo Vitti, 1 and Massimo Tonacchera 1 Loss-of-function mutations of the thyrotropin receptor (TSHr) may be responsible for congenital hypothyroidism or isolated hyperthyreotropinemia. To study cell surface expression of inactivating TSHr mutations detected in patients with isolated hyperthyreotropinemia (L252P, Q8fsX62, P27T, E34K, R46P, D403N, W488R, and M527T), we used the Agilent 2100 bioanalyzer to perform microchip flow cytometry analysis. The previously described TSHr inactivating mutation T477I was used as control. The level of receptor expression in COS-7 cells transfected with the T477I measured by binding assay was four times lower with respect to the wild-type TSHr. The very low expression of T477I was confirmed by fluorescence-activated cell sorting (FACS) analysis and by microchip flow cytometry analysis, suggesting that this method can be a reliable system to measure receptor cell surface expression. Other inactivating TSHr mutations were expressed in COS-7 cells for binding studies, FACS analysis, and microchip flow cytometry analysis. Binding studies showed that L252P, Q8fsX62, P27T, E34K, R46P, D403N, W488R, and M527T mutants had a low expression at the cell surface, as demonstrated by Bmax values. Data obtained by binding studies were in good agreement with data obtained by FACS analysis and microchip flow cytometry analysis. In conclusion, the low number of cells required for analysis and the ease of use make the microchip flow cytometry analysis a very reliable and favorable system to study cell surface expression of TSHr mutations. Introduction G -protein-coupled receptors (GPCRs) constitute a large family of proteins whose primary function is to transduce extracellular stimuli into intracellular signals. Many GPCRs have a certain basal activity (constitutive ac- tivity) and thus can activate G proteins in the absence of the agonist (1,2). Interestingly, it has been encountered that mu- tations of these receptors are able to dramatically increase this constitutive agonist-independent receptor activity (3) or in- hibit ligand-induced activation (4). Gain- and loss-of-function mutations in genes encoding GPCRs have been identified as a cause of human diseases (5). Thyroid-stimulating hormone (TSH) receptor, together with the follicle-stimulating hormone (FSH) and the lutei- nizing hormone (LH) receptors, is a member of a subfamily of seven transmembrane GPCRs. Inactivating mutations of the TSH receptor have been described in patients with congenital hypothyroidism and a normal or hypoplastic eutopic thyroid gland or isolated hyperthyreotropinemia (4,6–8). The func- tional activity of wild type and inactivating TSH recep- tor mutations are usually studied in eukaryotic cells after transient expression. Inactivating mutations are usually en- trapped intracellularly and have a defective expression at cell surface (6). Cell surface expression can be measured by dif- ferent methodologies, such as binding experiments or FACS analysis (9). A new procedure has been introduced to detect cell sur- face and intracellular protein targets by antibody staining. The 2100 bioanalyzer was introduced by Agilent Technologies as the first commercially available microfluidic analysis sys- tem for the electrophoretic separation and analysis of DNA, RNA, and proteins. With the introduction of the cell assay extension, the instrument is capable of measuring fluorescent- labeled cells based on simple flow cytometric analysis (10). Typical cell applications of this instrument are apoptosis de- tection, determination of transfection efficiency, and moni- toring protein expression by antibody staining. The Agilent 2100 bioanalyzer is capable of two-color fluorescence detec- tion and runs disposable microfluidic glass chips (Fig. 1). This 1 Dipartimento di Endocrinologia e Metabolismo, Centro Eccellenza AmbiSEN, Universita ` di Pisa, Pisa, Italy. 2 Divisione di Anatomia Patologica, Dipartimento di Oncologia, Universita ` di Pisa, Pisa, Italy. THYROID Volume 17, Number 9, 2007 ª Mary Ann Liebert, Inc. DOI: 10.1089=thy.2007.0114 861