Cloning and characterization of the human soluble adenylyl cyclase

Weidong Geng, Zenglu Wang, Jianning Zhang, Berenice Y. Reed, Charles Y C Pak, Orson W. Moe

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Abstract

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. Cytqplasmic 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. Mg2+, Mn2+, and Ca2+ all increased adenylyl cyclase activity in a dose-dependent manner. While hsAC had a minimal response to HCO3- in the absence of divalent cations, HCO3- robustly stimulated Mg2+-bound hsAC but inhibited Mn2+-bound hsAC in a dose-dependent manner. In summary, hsAC is a divalent cation and HCO 3- sensor, and its HCO3- sensitivity is modulated by divalent cations.

Original languageEnglish (US)
Pages (from-to)C1305-C1316
JournalAmerican Journal of Physiology - Cell Physiology
Volume288
Issue number6 57-6
DOIs
StatePublished - Jun 1 2005

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Keywords

  • Bicarbonate sensor
  • Calcium homeostasis
  • Hypercalciuria

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

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