The cloning of a Caenorhabditis elegans guanylyl cyclase and the construction of a ligand-sensitive mammalian/nematode chimeric receptor.
Substantial guanylyl cyclase activity was detected in membrane fractions prepared from Caenorhabditis elegans (100 pmol cGMP/min/mg at 20 degrees C or 500 pmol cGMP/min/mg at 37 degrees C), suggesting the potential existence of orphan cyclase receptors in the nematode. Using degenerate primers, a cDNA clone encoding a putative membrane form of the enzyme (GCY-X1) was obtained. The apparent cyclase was most closely related to the mammalian natriuretic peptide receptor family, and retained cysteine residues conserved within the extracellular domain of the mammalian receptors. Expression of the cDNA in COS-7 cells resulted in low, but detectable guanylyl cyclase activity (about 2-fold above vector alone). The extracellular and protein kinase homology domain of the mammalian receptor (GC-B) for C-type natriuretic peptide (CNP) was fused to the catalytic domain of GCY-X1 and expressed in COS-7 cells to determine whether ligand-dependent regulation would now be obtained. The resulting chimeric protein (GC-BX1) was active, and CNP elevated cGMP in a concentration-dependent manner. Subsequently, a search of the genome data base demonstrated the existence of at least 29 different genes from C. elegans that align closely with the catalytic domain of GCY-X1, and thus an equally large number of different regulatory ligands may exist.
Baude
E J
EJ
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas, 75235-9050, USA.
Arora
V K
VK
Yu
S
S
Garbers
D L
DL
Wedel
B J
BJ
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D017173:Caenorhabditis elegans / Q000201:enzymology*; D029742:Caenorhabditis elegans Proteins; D003001:Cloning, Molecular; D006152:Cyclic GMP / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry; D006162:Guanylate Cyclase / Q000737:chemistry / Q000235:genetics*; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017461:Receptors, Atrial Natriuretic Factor / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins; D017422:Sequence Analysis, DNA
D029742:Caenorhabditis elegans Proteins; D018076:DNA, Complementary; D008024:Ligands; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C106702:GCY-28 protein, C elegans; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; D006152:Cyclic GMP