INTERAZIONE CALCIUM-SENSING RECEPTOR-FILAMINA A E CARATTERIZZAZIONE DEL PATHWAY DI TRASDUZIONE DEL SEGNALE

Background: Calcium sensing receptor (CaSR) is a seven-transmembrane domains G-protein coupled receptor, with a large extracellular domain and a intracellular tail. It is important for maintaining the calcium steady state. In fact, it is able to detect variations in the extracellular calcium concentration ([Ca2+]o) and to activate a complex signal transduction pathway that causes increase of intracellular levels of inositol 1,4,5-trisphosphate (IP3) and calcium, activation of PKC and of MAPK cascade. The MAPK activation requires CaSR interaction with the scaffold protein Filamin A (FLNA). Just inside the binding region with the FLNA there is a polymorphism of the receptor tail, R990G, that induces a gain of function in the activity of the receptor, increasing the sensitivity to the calcimimetic R-568 and inducing different signal propagation compared to the WT CaSR. Moreover, both CaSR and FLNA are involved in tumors of the parathyroid glands and it has been demonstrated that CaSR activity is down-regulated in these tumors. The aim of this thesis is to confirm the hypothesis that the wild-type and polymorphic CaSR follow two different signal transduction pathways and to investigate the involvement of FLNA in this process. Therefore, we investigate the interaction CaSR-FLNA in parathyroid adenomas and carcinomas. Materials and Methods: FLNA was silenced with a Short interfering RNA in renal embryonic HEK-293 cells transfected with WT or polymorphic CaSR. The CaSR activity was assessed by measuring the activity of ERK 1/2 by Western blot. The expression of FLNA and CaSR were evaluated by immunofluorescence in HEK-293 cells and by immunofluorescence and immunohistochemistry in parathyroid adenomas and carcinomas. CaSR and FLNA mRNA levels were measured with Real-Time PCR in HEK-293 cells and in 30 parathyroid adenomas; parathyroid adenomas of the same samples were genotyped for the SNP R990G using the technique Taqman genotyping assay. Results: Western Blot data showed a decrease of expression and activity in both forms of the receptor (WT CaSR and R990G CaSR) in the absence of FLNA. Real-Time results of HEK-293 cells show a decrease of mRNA expression in both WT CaSR (0.55±0.23 vs 1.56± 0.80, p-value=0.07) that R990G CaSR (0.51±0.37 vs 1.82±1.43, p-value=0.41) in the absence of FLNA compared to the control. The presence of the calcimimetic R-568 seems to mask the differences in the absence of FLNA, keeping the gain of function of R990G CaSR. The CaSR and FLNA resulted down-regulated in parathyroid glands tumors. Immunohistochemistry of parathyroid adenomas and carcinomas showed a decrease of FLNA expression related to the degree of tumor malignancy. Furthermore, the correlation between CaSR and FLNA mRNA levels and R990G SNP in 30 adenomas showed a significant rise of mRNA expression of both CaSR type (n=28 AA, 1.07±0.97; n=2 AG, 3.39±1.16, p-value=0.003) and FLNA (n=28 AA, 0.14±0.08; n=2 AG, 3.35±0.09, p-value=0.002) in the presence of the heterozygous genotype AG. Conclusion: this study has allowed us to show that FLNA is required for the CaSR-inducted stimulation of ERK1/2 activity, both in WT CaSR and R990G CaSR. So we could hypothesize that the gain of function of the CaSR R990G could be due to a stronger interaction with FLNA, induced by non-conservative polymorphism in the binding region for FLNA. Furthermore, we can conclude that the CaSR and FLNA interaction is also crucial in parathyroid tumors, in which we demonstrated a positive correlation of two proteins expression, both down-regulated in tumors. The correlation of FLNA and CaSR mRNA expression with R990G polymorphism showed the increase of both proteins expression in the presence of the polymorphic variant, suggesting a protective role in the parathyroid glands tumors, with unknown mechanism.