STRUCTURAL STUDIES OF ATRIAL NATRIURETIC FACTOR RECEPTOR

心房利钠因子（atrial natriuretic factor，ANF）是新近发现的一种肽类激素 从心房排出，心房有强的利钠和血管舒张剂 活动 该激素的利钠利尿作用似乎不涉及 仅其对肾内血流动力学和肾小管的直接影响 Na+重吸收以及对肾上腺醛固酮和 垂体加压素释放 ANF对靶器官的作用是 通过特定的细胞膜受体介导，ANF结合到 引起细胞内cGMP的刺激和cAMP的抑制。 的 心钠素受体、核苷酸环化酶和细胞凋亡的机制关系 生物学效应尚不清楚。 本建议的总体目标是 ANF受体的最终目的是 阐明心钠素及其受体作用的分子机制。 ANF受体的纯化将在洗涤剂洗涤后进行 通过1）使用ANF类似物作为亲和层析的增溶， 配体，2）来自与ANF的阳离子交换的“亲和洗脱”，3） 抗ANF抗体对ANF交联受体免疫吸附，或4） 使用针对结合位点产生的抗体的免疫亲和层析 上述肽。 最终纯化将通过 常规液相色谱法的组合。 纯化的ANF 受体将用于表征、抗体生产、肽 映射、部分序列测定和可能的重构 问题研究 ANF受体的结合位点结构将使用 新设计的“顺序亲和标记”方案。 ANF类似物， 共价连接的亲电体将被允许与受体结合， 其中亲电反应是不利的，但 绑定不受影响。 然后将条件转移到有利于 反应完成交联。 通过选择亲电试剂和 连接位置，具有高交联的亲和标记试剂 效率和残留物特异性。 这些试剂将 用于共价标记，用于结合位点的制备分离 肽。 标记的肽将在BrCN裂解后通过 使用抗ANF抗体的免疫亲和层析，随后进行反向 相HPLC。 将确定其氨基酸序列。 抗体 针对结合位点的肽将使用合成肽产生， 阐明的序列。 抗体抑制ANF结合的能力 将测量这些序列对受体的作用，以研究这些序列在 或围绕ANF结合位点。

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.