Atrial natriuretic factor (ANF) is a recently discovered peptide hormone from the heart atrium which has potent natriuretic and vasorelaxant activities. The natriuretic effect of the hormone appears to involve not only its direct effect on intrarenal hemodynamics and tubular Na+-reabsorption but also its inhibitory effects on adrenal aldosterone and pituitary vasopressin release. The action of ANF at the target organs is mediated through specific cell membrane receptors, ANF binding to which causes intracellular stimulation of cGMP and suppression of cAMP. The mechanistic relationship of ANF receptor, nucleotide cyclases and the biological effects are unknown. The general objective of this proposal is the structural characterization of ANF receptors with an ultimate purpose of elucidating the molecular mechanism of ANF and ANF receptor action. Purification of ANF receptor will be carried out after detergent solubilization by 1) affinity chromatography using an ANF analog as a ligand, 2) """"""""affinity elusion"""""""" from cation-exchange with ANF, 3) immuno-adsorption of ANF-crosslinked receptor by anti ANF antibody, or 4) immunoaffinity chromatography using antibody raised against binding site peptide described above. Final purification will be achieved by a combination of conventional liquid chromatography. The purified ANF receptor will be used for characterization, antibody production, peptide mapping, partial sequence determination and possibly for reconstitution studies. The binding site structure of ANF receptor will be probed using a newly designed """"""""sequential affinity labeling"""""""" protocol. ANF analog with a covalently attached electrophile will be allowed to bind to receptor under the conditions where the electrophilic reaction is unfavored but the binding not affected. The conditions are then shifted to favor the reaction to complete crosslinking. By selecting the electrophile and position of attachment, affinity labeling reagents with high crosslinking efficiency and residue-specificity will be prepared. Such reagents will be used for covalent labeling for preparative isolation of binding site peptide. The labeled peptide will be isolated after BrCN cleavage by immunoaffinity chromatography using anti-ANF antibody followed by reverse phase HPLC. Their amino acid sequences will be determined. Antibodies against binding site peptides will be raised using systhetic peptides with the elucidated sequences. Ability of the antibodies to inhibit ANF binding to the receptor will be measured to study arrangement of these sequences in or around the ANF binding site.
