A fast method to detect cell surface expression of thyrotropin receptor (TSHr): the microchip flow cytometry analysis.
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.
Agretti
Patrizia
P
Dipartimento di Endocrinologia e Metabolismo, Centro Eccellenza AmbiSEN, Università di Pisa, Pisa, Italy.
De Marco
Giuseppina
G
Capodanno
Alessandra
A
Ferrarini
Eleonora
E
Dimida
Antonio
A
Sansone
Daniela
D
Collecchi
Paola
P
Pinchera
Aldo
A
Vitti
Paolo
P
Tonacchera
Massimo
M
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D046208:Microchip Analytical Procedures / Q000379:methods*; D009154:Mutation; D011989:Receptors, Thyrotropin / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics
D011989:Receptors, Thyrotropin