Background: To evaluate some aspects of renal CaSR physiopathology, we analyzed the signaling underling two constitutively active variants of the CaSR. Methods: Constructs encoding human wild-type CaSR (hCaSR-wt) and its constitutively active (hCaSR-R990G; hCaSR-N124K) and inactive variants (hCaSR-del121) were transiently transfected in HEK cells. Results: Immunofluorescence studies revealed that both hCaSR-wt and its activating variants were expressed at the plasma membrane, whereas the inactive form localized intracellularly. The physiological agonist, calcium (Ca++ 5mM), and the calcimimetic NPS-R568 (5μM) induced a significant increase in cytosolic calcium in cells expressing hCaSR-wt and its active variants, compared to mock. We also found that the basal intracellular calcium was significantly lower in cells expressing hCaSR-wt and its activating variants compared to mock and hCaSR-del121 transfected cells. Low calcium levels are expected to make cells more sensitive to intracellular calcium changes in response to CaSR agonists. In line, FRET studies using D1ER probe, which detects [Ca++]ER directly demonstrated a significant higher calcium accumulation in cells expressing the activating CaSR variants. Since the storage of calcium in the ER is mainly regulated by SERCA, the activity and the expression of this pump were evaluated. Compared to hCaSR-wt expressing cells SERCA expression and activity were found significantly increased in cells expressing activating CaSR variants. An inverse correlation with PMCA was also found. Conclusions: Together, these findings indicate that for the efficiency of calcium signaling system, cells monitor cytosolic and ER calcium levels regulating the expression of the SERCA and PMCA. To our knowledge this is the first demonstration that a complex parallel adaptive feedback can explain the molecular basis of constitutively active variants of the CaSR. 

New Regulatory Mechanism Explaining the Molecular Basis of Constitutively Active Variants of the Calcium-Sensing Receptor (CaSR)

RANIERI, MARIANNA;TAMMA, GRAZIA;Di Mise A;SVELTO, Maria;VALENTI, Giovanna
2012-01-01

Abstract

Background: To evaluate some aspects of renal CaSR physiopathology, we analyzed the signaling underling two constitutively active variants of the CaSR. Methods: Constructs encoding human wild-type CaSR (hCaSR-wt) and its constitutively active (hCaSR-R990G; hCaSR-N124K) and inactive variants (hCaSR-del121) were transiently transfected in HEK cells. Results: Immunofluorescence studies revealed that both hCaSR-wt and its activating variants were expressed at the plasma membrane, whereas the inactive form localized intracellularly. The physiological agonist, calcium (Ca++ 5mM), and the calcimimetic NPS-R568 (5μM) induced a significant increase in cytosolic calcium in cells expressing hCaSR-wt and its active variants, compared to mock. We also found that the basal intracellular calcium was significantly lower in cells expressing hCaSR-wt and its activating variants compared to mock and hCaSR-del121 transfected cells. Low calcium levels are expected to make cells more sensitive to intracellular calcium changes in response to CaSR agonists. In line, FRET studies using D1ER probe, which detects [Ca++]ER directly demonstrated a significant higher calcium accumulation in cells expressing the activating CaSR variants. Since the storage of calcium in the ER is mainly regulated by SERCA, the activity and the expression of this pump were evaluated. Compared to hCaSR-wt expressing cells SERCA expression and activity were found significantly increased in cells expressing activating CaSR variants. An inverse correlation with PMCA was also found. Conclusions: Together, these findings indicate that for the efficiency of calcium signaling system, cells monitor cytosolic and ER calcium levels regulating the expression of the SERCA and PMCA. To our knowledge this is the first demonstration that a complex parallel adaptive feedback can explain the molecular basis of constitutively active variants of the CaSR. 
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11586/65345
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