The guanylyl cyclase-A receptor transduces an atrial natriuretic peptide/ATP activation signal in the absence of other proteins.
Attempts to activate partially purified preparations of the guanylyl cyclase-A (GC-A) receptor with atrial natriuretic peptide (ANP) have previously failed, leading to speculation that essential cofactors are lost during purification procedures. The receptor was modified to contain the FLAG epitope (DYKDDDDK), expressed in Sf9 cells, and purified to apparent homogeneity (4.3 mumol cyclic GMP formed/min/mg protein; 5.8 mmol 125I-ANP binding site/mg protein) by a combination of immunoaffinity, Q-Sepharose FF, and wheat germ agglutinin batch chromatography. High initial protein/detergent ratios, the presence of glycerol (40%), and the inclusion of protein phosphatase inhibitors in all buffers resulted in the purification of a receptor that continued to transduce the ANP/ATP activation signal. Both native and purified GC-A contained a single class of high affinity ANP binding sites (Kd = 60 pM) and an equivalent EC50 for ATP (0.3 mM). Positive cooperativity as a function of MnGTP was retained during purification. Thus, GC-A is capable of transducing a ligand binding signal in the absence of other proteins.
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235, USA.
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Wong
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Foster
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Chen
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Garbers
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism / Q000494:pharmacology*; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006162:Guanylate Cyclase / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010455:Peptides; D017461:Receptors, Atrial Natriuretic Factor; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000302:isolation & purification / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction; D018411:Spodoptera; D014162:Transfection
D000939:Epitopes; D009842:Oligopeptides; D010455:Peptides; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C029273:P(3)-1-(2-nitro)phenylethyladenosine 5'-triphosphate; D009320:Atrial Natriuretic Factor; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; C085706:FLAG peptide; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; C091715:atrial natriuretic factor receptor A