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Functional Co-localization of Transfected Ca2+-stimulable Adenylyl Cyclases with Capacitative Ca2+ Entry Sites

1996; Elsevier BV; Volume: 271; Issue: 21 Linguagem: Inglês

10.1074/jbc.271.21.12438

1083-351X

Kent A. Fagan, Rajesh Mahey, Dermot M.F. Cooper,

Receptor Mechanisms and Signaling

Three adenylyl cyclases (ACI, ACIII, and ACVIII) have been described, which are putatively Ca2+-stimulable, based on in vitro assays. However, it is not clear that these enzymes can be regulated by physiological rises in [Ca2+]i when expressed in intact cells. Furthermore, it is not known whether transfected adenylyl cyclases might display the strict requirement for capacitative Ca2+ entry that is shown by the Ca2+-inhibitable ACVI, which is indigenous to C6-2B glioma cells (Chiono, M., Mahey, R., Tate, G., and Cooper, D. M. F.(1995) J. Biol. Chem. 270, 1149-1155). In the present study, ACI, ACIII, and ACVIII were heterologously expressed in HEK 293 cells, and conditions were devised that distinguished capacitative Ca2+ entry from both internal release and nonspecific elevation in [Ca2+]i around the plasma membrane. Remarkably, not only were ACI and ACVIII largely insensitive to Ca2+ release from stores, but they were robustly stimulated only by capacitative Ca2+ entry and not at all by a substantial increase in [Ca2+]i at the plasma membrane elicited by ionophore. (ACIII, reflecting its feeble in vitro sensitivity to Ca2+, was unaffected by any [Ca2+]i rise.) These results suggest a quite unsuspected, essential association of Ca2+-sensitive adenylyl cyclases with capacitative Ca2+ entry sites, even when expressed heterologously. Three adenylyl cyclases (ACI, ACIII, and ACVIII) have been described, which are putatively Ca2+-stimulable, based on in vitro assays. However, it is not clear that these enzymes can be regulated by physiological rises in [Ca2+]i when expressed in intact cells. Furthermore, it is not known whether transfected adenylyl cyclases might display the strict requirement for capacitative Ca2+ entry that is shown by the Ca2+-inhibitable ACVI, which is indigenous to C6-2B glioma cells (Chiono, M., Mahey, R., Tate, G., and Cooper, D. M. F.(1995) J. Biol. Chem. 270, 1149-1155). In the present study, ACI, ACIII, and ACVIII were heterologously expressed in HEK 293 cells, and conditions were devised that distinguished capacitative Ca2+ entry from both internal release and nonspecific elevation in [Ca2+]i around the plasma membrane. Remarkably, not only were ACI and ACVIII largely insensitive to Ca2+ release from stores, but they were robustly stimulated only by capacitative Ca2+ entry and not at all by a substantial increase in [Ca2+]i at the plasma membrane elicited by ionophore. (ACIII, reflecting its feeble in vitro sensitivity to Ca2+, was unaffected by any [Ca2+]i rise.) These results suggest a quite unsuspected, essential association of Ca2+-sensitive adenylyl cyclases with capacitative Ca2+ entry sites, even when expressed heterologously.