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 c yclase-A (GC-A) receptor with atrial natriuretic peptide (ANP) have pr eviously failed, leading to speculation that essential cofactors are l ost during purification procedures. The receptor was modified to conta in the FLAG epitope (DYKDDDDK), expressed in Sf9 cells, and purified t o apparent homogeneity (4.3 mu mol cyclic GMP formed/min/mg protein; 5 .8 nmol I-125-ANP binding site/mg protein) by a combination of immunoa ffinity, Q-Sepharose FF, and wheat germ agglutinin batch chromatograph y. 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 transduc e the ANP/ATP activation signal. Both native and purified GC-A contain ed a single class of high affinity ANP binding sites (K-d = 60 pM) and an equivalent EC(50) for ATP (0.3 mM). Positive cooperativity as a fu nction of MnGTP was retained during purification. Thus, GC-A is capabl e of transducing a ligand binding signal in the absence of other prote ins.