Cloning and characterization of the human soluble adenylyl cyclase.
We identified the human ortholog of soluble adenylyl cyclase (hsAC) in a locus linked to familial absorptive hypercalciuria and cloned it from a human cDNA library. hsAC transcripts were expressed in multiple tissues using RT-PCR and RNA blotting. RNA blot analysis revealed a predominant 5.1-kb band in a multiple human tissue blot, but three splice transcript variants were detected using RT-PCR and confirmed by performing sequence analysis. Immunoblot analysis showed 190- and 80-kDa bands in multiple human cell lines from gut, renal, and bone origins in both cytosol and membrane fractions, including Caco-2 colorectal adenocarcinomas, HEK-293 cells, HOS cells, and primary human osteoblasts, as well as in vitro induced osteoclast-like cells. The specificity of the antiserum was verified by peptide blocking and reduction using sequence-specific small interfering RNA. Confocal immunofluorescence cytochemistry localized hsAC primarily in cytoplasm, but some labeling was observed in the nucleus and the plasma membrane. Cytoplasmic hsAC colocalized with microtubules but not with microfilaments. To test the function of hsAC, four constructs containing catalytic domains I and II (aa 1-802), catalytic domain II (aa 231-802), noncatalytic domain (aa 648-1,610), and full-length protein (aa 1-1,610) were expressed in Sf9 insect cells. Only catalytic domains I and II or full-length proteins showed adenylyl cyclase activity. Mg(2+), Mn(2+), and Ca(2+) all increased adenylyl cyclase activity in a dose-dependent manner. While hsAC had a minimal response to HCO(3)(-) in the absence of divalent cations, HCO(3)(-) robustly stimulated Mg(2+)-bound hsAC but inhibited Mn(2+)-bound hsAC in a dose-dependent manner. In summary, hsAC is a divalent cation and HCO(3)(-) sensor, and its HCO(3)(-) sensitivity is modulated by divalent cations.
Geng
Weidong
W
Center for Mineral Metabolism and Clinical Research, Department of Internal Medicine. University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75390-8885, USA.
Wang
Zenglu
Z
Zhang
Jianning
J
Reed
Berenice Y
BY
Pak
Charles Y C
CY
Moe
Orson W
OW
D000262:Adenylyl Cyclases / Q000096:biosynthesis / Q000502:physiology*; D001483:Base Sequence; D001639:Bicarbonates / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data
D001639:Bicarbonates; D008345:Manganese; D000262:Adenylyl Cyclases; D008274:Magnesium