Endocrine Abstracts

In two siblings suffering from congenital hypothyroidism we identified a homozygous missence mutation Ala579Val in transmembrane helix 5 of the thyrotropin receptor (TSHR) gene which motivated us to investigate molecular details of this mutation.

We were interested, firstly, in the functional effects regarding signal transduction and, secondly, in the particular structural properties of the wild type receptor and the Ala579Val mutant. The aim was to gain deeper mechanistic insights into the inactivation resulting from mutations at this position and to understand the biochemical properties of other potentially participating amino acids. We analysed structural-functional relationships of the TSHR by molecular modelling using the latest crystal structures of GPCRs as templates for TSHR homology models and we designed and functionally tested side chain mutations at this position to Ser, Gln, Phe, Met and Leu by determination of b-TSH induced intracellular cAMP formation.

The functional characterization of the patients’ mutation revealed a complete loss of function. Moreover side chain mutations to Leu, Met, and Gln at this position resulted in a complete or partial loss of signalling capability. By contrast, substitutions to Ser and Phe were behaved like the wild type protein. Investigations of TSHR homology models suggest, that substitution of the small Ala to a more bulky side chain causes a signalling incompetent receptor conformation. Especially side chains that are branched at the beta-carbon are structurally not tolerated and lead to completely impaired signalling.

In summary, investigation of this newly identified mutation, Ala579Val, of the TSHR helps to explain functional properties as well as structural changes caused by the mutation. Our findings provide significant structural and functional implications for mechanisms of signal transduction of the TSHR and homologous glycoprotein-hormone receptors.