Forrest Fuller$, J. Gordon Porter, Ann E. Arfsten, Judy Miller, James W. Schilling,. Robert M. Scarborough, John A. Lewicki, and Dale B. Schenk. From California ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 263,No. 19, Issue of July 5 , pp. 9395-9401,1988 Prmted u1 U.S.A.
0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.
Atrial Natriuretic Peptide Clearance Receptor COMPLETE SEQUENCE AND FUNCTIONAL EXPRESSION OF cDNA CLONES* (Received for publication, November 12, 1987)
Forrest Fuller$,J. Gordon Porter, Ann E. Arfsten, Judy Miller, James W. Schilling, Robert M. Scarborough, JohnA. Lewicki, and DaleB. Schenk From California Biotechnology,Inc., Mountain View,California 94043 The major class of atrial natriuretic peptide (ANP) have identified at least two ANP-binding proteins with M, receptors was isolated from cultured vascular smooth -130,000 and -60,000 as determined by SDS-PAGE analysis muscle cells, and a partial amino acid sequence was under reducing conditions (11, 15-28). Furthermore, a funcobtained. This allowed the isolation of cDNA clones tionaldistinction between receptor types is suggested by from which theentire amino acid sequencewas estab- pharmacological studies. Whereas an important receptor-melished. The smooth muscle cell ANP receptor appears diated effect of ANP is the stimulation of particulate guanylto be synthesized as a 537-amino acid precursorwith an N-terminal membrane translocation signal. The ma- ate cyclase activity (29,30), several truncated analogs of ANP ture form consists of 496 amino acids with a single which neither stimulate guanylate cyclase nor antagonize the potentialtransmembranedomainpredictinga 37- ANP-mediated stimulationof guanylate cyclase are nevertheaminoacidcytoplasmicdomainanda large, acidic, less able to bind with high affinity to the major class of extracellulardomain low in cysteine and probably con- receptors observed on vascular smooth muscle cells (29-33). is there- Cross-linking studies in vascular endothelial cells suggest that taining attached carbohydrate. The receptor fore similar in structure to the growth factor receptorssuch ANP analogs bind specifically to the -60-kDa subunit but notably lacksrepetitive cysteine-rich domains and (30),’ whereas only analogs which activate guanylate cyclase has arelatively small intracellular domain. Expressionappear to be able to bind to the-130-kDa subunit (11,16,17, of the cloned receptor Xenopus in oocytes elicited high 28). Takayanagi et al. (34) have recently purified two strucaffinity, membrane-associated binding sites for ANP turally dissimilar (35) ANP binding activities from bovine and for truncated and internally deleted analogs of adrenal membranes to near homogeneity and have demonANP. These results reflect the ligand binding specificstrated that guanylate cyclase activity is exclusively associity foundforthemajor class ofANPreceptorson smooth muscle cells and thus provide additional evi- ated with an -135-kDa polypeptide, whereas truncated ANP dence thattwo distinct ANP receptorsexist since ANP analog binding is exclusively associated with an -62-kDa receptor-coupled guanylatecyclase activity exhibits a polypeptide. These observations clearly indicate that functionally and very different ANP analogspecificity. structurally distinct ANPreceptors exist. However, the function and physiological role of the receptor with the -60-kDa binding subunit have not been elucidated. Recently, Maack Atrial natriuretic peptide (ANP),’ a hormone which is synthesized and secreted by the atria of the heart, evokes a et al. (36) have proposed a novel function for this receptor variety of physiological responses affecting cardiovascular based on the observation that in vivo administration of an homeostasis. These effects which collectivelylead to reduction internally deleted ANP analog results in an increase in cirin blood volume include diuresis and natriuresis (1-3), inhi- culating ANP levels. They suggest that theprimary function bition of aldosterone secretion (4, 51, and vasorelaxation (6). of this receptor is sequestration and metabolic clearance of Such diverse effects are reflected in the wide variety of cell ANP and have therefore designated it as the C-receptor. As types and target tissues on which specific binding sites for an initial step toward further defining receptor-mediated ANP have been identified (7-17) and in the varied responses functions of ANP and determining the molecular structures of the different ANP receptors, the major ANP receptor from of these cells and tissues to ANP. Diversity of function is also indicated by the observed cultured bovine aortic smooth muscle (BASM) cells was puheterogeneity in ANP-binding sites. Cross-linking studies rified to apparenthomogeneity with functional binding activity (37). We now report the direct determination of the N* The costs of publication of this article were defrayed in part by terminal amino acid sequence of this proteinand theisolation the payment of page charges. This article must therefore be hereby of corresponding cDNA clones. That the clones encode the marked “advertisement” in accordance with 18 U.S.C. Section 1734 ANP C-receptor found on BASM cells is demonstrated by the solely to indicate this fact. The nucleotide sequence(s)reported in thispaper has been submitted findings that the molecular weight and structural characterto the GenBankTM/EMBLData Bank with accession number(s) istics of the encoded polypeptide, the cell and tissue sources 503876. of homologous mRNAs, and the ANP binding imparted on $ TOwhom correspondence should be addressed California Biomembranes from Xenopus oocytes injected with the cloned technology, Inc., 2450 Bayshore Pkwy., Mountain View, CA 94043. The abbreviations used are: ANP, atrial natriureticpeptide; ANP sequence all reflect the known properties of the ANP C C-receptor, ANP clearance receptor; rANP, rat ANP; SDS-PAGE, receptor. sodium dodecyl sulfate-polyacrylamide gel electrophoresis; BASM, bovine aortic smooth muscle; PTH, phenylthiohydantoin; HPLC, octaethylene glycol high performance liquid chromatography; CI2E~, dodecyl ether; kb, kilobase.
F. Fuller, J. G . Porter, A. E. Arfsten, J. Miller, J. W. Schilling, R. M. Scarborough, J. A. Lewicki, and D. B. Schenk, unpublished observations.
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A N P C-receptor cDNA Sequence and Expression
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