Site-directed mutational analysis of a membrane guanylate cyclase cDNA reveals the atrial natriuretic factor signaling site.
Natriuretic peptides are structurally related hormones that regulate hemodynamics of the physiological processes of diuresis, water balance, and blood pressure. One of the second messengers of these hormones is cGMP, and the type of receptor that is involved in the generation of cGMP is also a guanylate cyclase. Recent genetic evidence has revealed such a receptor family; two family members, GC-A and GC-B, have been cloned. We now describe the molecular cloning, sequencing, and expression of a cDNA clone from rat adrenal gland that encodes a membrane guanylate cyclase, GC alpha, that, with the exception of two amino acids, is structurally identical to GC-A and conforms to the purported topographical model of GC-A. The two amino acid changes are the substitutions Gln338----His338 and Leu364----Pro364, involving single nucleotide changes, CAG----CAC and CTG----CCG, respectively. Expression studies indicate that GC alpha cyclase activity is independent of the known natriuretic peptides, and direct binding studies demonstrate that GC alpha is not an ANF receptor. To determine the importance of Gln338 and Leu364 in ANF signaling, the GC alpha cDNA regions encoding amino acid residues 338 and 364 were remodeled by oligonucleotide-directed mutagenesis. A double mutant encoding Gln338 and Leu364, and a single-substitution mutant encoding Leu364 expressed both ANF binding and ANF-dependent cyclase activities, but the mutant encoding Gln338 and a deletion mutant lacking residue 364 did not express either of the above activities. These results define the critical role of Leu364 in ANF signal transduction.
Section of Regulatory Biology, Cleveland Clinic Foundation, OH 44195-5068.
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D000595:Amino Acid Sequence; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D003001:Cloning, Molecular; D015723:Gene Library; D006162:Guanylate Cyclase / Q000235:genetics* / Q000502:physiology; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D051381:Rats; D017461:Receptors, Atrial Natriuretic Factor; D011956:Receptors, Cell Surface / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
D015345:Oligonucleotide Probes; D011956:Receptors, Cell Surface; D009320:Atrial Natriuretic Factor; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor