Abstract
A variant of the alpha 2 subunit of soluble guanylyl cyclase (alpha 2i) containing 31 additional amino acids was identified in a number of cell lines and tissues. The in-frame sequence of the insert was within the proposed catalytic domain of guanylyl cyclases and was homologous to a region within the putative catalytic domain of adenylyl cyclases. Messenger RNA for the new variant was detected in some but not all cell lines and tissues expressing the alpha 2 subunit. The novel form, as well as the alpha 2 subunit lacking the insert, were coexpressed with the beta 1 subunit in Sf9 and COS-7 cells; alpha 2/beta 1 coexpression yielded a NO-sensitive recombinant protein, whereas the coexpressed alpha 2i/beta 1 subunits exhibited no guanylyl or adenylyl cyclase activities. Because both subunits (alpha 2i/beta 1) copurified, the novel variant retains its ability to heterodimerize. In coexpression experiments, the alpha 2i subunit competed with the alpha 2 subunit for dimerization with the beta 1 subunit, thereby reducing alpha 2/beta 1-catalyzed guanylyl cyclase activity. These data show that the novel variant functions as a dominant negative protein and that post-transcriptional mRNA processing represents a potential mechanism for regulation of NO-sensitive guanylyl cyclase activity.
Highlights
The mechanisms by which the soluble or membrane forms of guanylyl cyclase are regulated have been explored at both the protein and the gene level
Materials—2,2-Diethyl-1-nitroso-oxyhydrazine sodium salt (DEANO)1 [24] was purchased from NCI Chemical Carcinogen Repository. 10-fold concentrated stock solutions were prepared in 10 mM NaOH. cDNA libraries were purchased from Stratagene or Clontech
A novel variant cDNA coding for a guanylyl cyclase subunit (␣2i) was obtained from different human cell lines using polymerase chain reaction (PCR) with degenerate primers based on conserved sequences of the catalytic domain of mammalian guanylyl cyclases
Summary
Materials—2,2-Diethyl-1-nitroso-oxyhydrazine sodium salt (DEANO)1 [24] was purchased from NCI Chemical Carcinogen Repository. 10-fold concentrated stock solutions were prepared in 10 mM NaOH. cDNA libraries were purchased from Stratagene (human fetal brain 936206, human T-cell 938200, human liver 937220, and human colon 937204) or Clontech (human endothelial cell HL1164b and human testis HL1010b). Expression of subunits in COS-7 cells and immunoblot analysis of the cytosolic proteins were performed as described [8]. The elution was performed with a linear 0 – 400 mM NaCl gradient collected in 5-ml fractions Both ␣2i and 1 subunits eluted at salt concentrations of approximately 200 mM NaCl, as monitored by immunoblotting. Nylyl cyclase activity of the obtained cytosolic fractions (80 –200 g/ assay tube) was determined by incubation for 15 min at 37 °C in the presence of 50 mM triethanolamine/HCl buffer, pH 7.4, containing 3 mM dithiotreitol, 1 mM 3-isobutyl-1-methylxanthine, 1 mM cyclic GMP, 5 mM creatine phosphate, 4.6 units/tube creatine phosphokinase, 0.5 mM [␣-32P]GTP (about 1 Ci), and 3 mM MgCl2 or 3 mM MnCl2, with or without 10 M DEA-NO in a total volume of 0.1 ml. Separation and purification of the enzyme-formed cAMP was as described [31]
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