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Atrial Natriuretic Peptide Induces Natriuretic PeptideReceptor-cGMP-dependent Protein Kinase Interaction*

Received for publication, April 18, 2003, and in revised form, July 1, 2003Published, JBC Papers in Press, July 10, 2003, DOI 10.1074/jbc.M304098200

Nathan Airhart, Yong-Feng Yang, Charles T. Roberts, Jr., and Michael Silberbach

From the Department of Pediatrics and the Heart Research Center, Oregon Health and Science University,Portland, Oregon 97239

Circulating natriuretic peptides such as atrial natri-uretic peptide (ANP) counterbalance the effects of hy-pertension and inhibit cardiac hypertrophy by activat-ing cGMP-dependent protein kinase (PKG). Natriureticpeptide binding to type I receptors (NPRA and NPRB)activates their intrinsic guanylyl cyclase activity, re-sulting in a rapid increase in cytosolic cGMP that sub-sequently activates PKG. Phosphorylation of the recep-tor by an unknown serine/threonine kinase is requiredbefore ligand binding can activate the cyclase. Whilesearching for downstream PKG partners using a yeasttwo-hybrid screen of a human heart cDNA library, weunexpectedly found an upstream association withNPRA. PKG is a serine/threonine kinase capable of phos-phorylating NPRA in vitro; however, regulation ofNPRA by PKG has not been previously reported. Herewe show that PKG is recruited to the plasma membranefollowing ANP treatment, an effect that can be blockedby pharmacological inhibition of PKG activation. Fur-thermore, PKG participates in a ligand-dependent gain-of-function loop that significantly increases the intrin-sic cyclase activity of the receptor. PKG translocation isANP-dependent but not nitric oxide-dependent. Our re-sults suggest that anchoring of PKG to NPRA is a keyevent after ligand binding that determines distal effects.As such, the NPRA-PKG association may represent anovel mechanism for compartmentation of cGMP-medi-ated signaling and regulation of receptor sensitivity.

The natriuretic peptides (NPs)1 are produced by the heart,the vasculature, and the kidney and are an ancient family ofpolypeptide hormones that regulate mammalian blood volumeand blood pressure. More recently, the ability of NPs to mod-ulate cell proliferation (1) and cardiac hypertrophy (2) has beendemonstrated. Physiologically important NP actions includenatriuresis (3), vasodilation (4), and ubiquitous inhibitory ac-tions such as inhibition of smooth muscle proliferation (1, 5),

cardiac fibroblast proliferation (6), cardiomyocyte hypertrophy(2, 79), sympathetic tone (10), renin-angiotensin-aldosteroneactivation (11), and hypothalamic-pituitary-adrenal axis sig-naling (1214). In disease states such as heart failure, NPactions may be limited by resistance to hormone effects (1517)that are at least in part because of insensitivity of the receptoritself (18).

Natriuretic peptide binding to type I receptors (NPRA andNPRB) on target cells activates their intrinsic guanylyl cyclaseactivity, resulting in a rapid increase in cGMP. DiffusiblecGMP acts as a second messenger primarily by stimulatingPKG (19). PKG is the major mediator of cGMP-induced smoothmuscle relaxation (20). Downstream NP effects that have beendirectly tied to activated PKG include modulation of the L-typecalcium channel (21, 22) and cross-talk with heterologous re-ceptors, such as G protein-coupled receptors (23, 24). Further-more, there is recent evidence that the membrane-bound gua-nylyl cyclase, NPRA, but not soluble cyclases that are activatedby nitric oxide, has potent effects on plasma membrane controlof the calcium ATPase pump (25), suggesting that NO- andNP-mediated effects are compartmentalized in cells.

Although the NPRA cDNA was first cloned more than 13years ago (26), its regulation remains poorly understood. In itsprebound state, the NPRA receptor exists as a homodimer (27),but ligand binding alone is insufficient to induce cyclase activ-ity. Rather, phosphorylation of six serine and threonine resi-dues in the intracellular juxtamembrane-kinase homology do-main makes the receptor susceptible to NP activation (28). Theprotein kinase that mediates receptor phosphorylation isunknown.

We have previously reported that ANP inhibits cardiac hy-pertrophy through cGMP/PKG-mediated activation of the ERKsignaling cascade at the level of MEK (9) but could not demon-strate a direct interaction between PKG and MEK. PKG sub-strates are membrane-bound (29), cytosolic (30), and intranu-clear (31). In an attempt to identify novel proteins that could becandidates for linking PKG to MEK, we used a cytosolic yeasttwo-hybrid system employing PKG as bait. We found that PKGdirectly interacts with NPRA. These results were initially quitesurprising, because PKG was previously thought to regulateonly downstream ANP targets. However, it has been demon-strated previously that PKG is a serine/threonine kinase capa-ble of phosphorylating NPRA in vitro (32). We report for thefirst time the regulation of NPRA by PKG.

EXPERIMENTAL PROCEDURES

Yeast Two-hybrid StudiesScreening for PKG-interacting proteinswas done using a commercially available system (CytoTrap, Strat-agene, La Jolla, CA) that identifies protein-protein interactions in theyeast cytoplasm. Rather than relying on transcriptional activation todetect interactions, the RAS signal transduction cascade is initiated byrecruitment of hSOS to the plasma membrane in a temperature-sensi-tive mutant yeast strain, cdc25H, by virtue of the interaction of its baitfusion partner with a myristoylated prey protein, which allows growth

* This work was supported by grants (to M. S.) from the Friends ofDoernbecher, the Dickinson Family Foundation, and the Glenn andJuanita Struble Research Fund of the Oregon Health and ScienceUniversity Heart Research Center. The costs of publication of thisarticle were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Doernbecher Chil-drens Hospital, 707 S.W. Gaines Rd., Mail Code CDRC-P, Portland, OR97239-2901. Tel.: 503-494-9899; Fax: 503-494-2824; E-mail: silberbm@ohsu.edu.

1 The abbreviations used are: NP, natriuretic peptide; PKG, cGMP-dependent protein kinase; NO, nitric oxide; ANP, atrial NP; PIPES,1,4-piperazinediethanesulfonic acid; SNP, S-nitroso-N-acetylpenicilla-mine; HEK, human embryonic kidney; MEK, mitogen-activated proteinkinase/extracellular signal-regulated kinase kinase; ERK, extracellularsignal-regulated kinase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 40, Issue of October 3, pp. 3869338698, 2003 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org 38693

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at 36 C. A bovine PKG I cDNA was cloned in-frame into the hSOSbait plasmid. A human heart cDNA library in the pMyr plasmid (Strat-agene) containing 7.4 106 independent clones was cotransfected withthe pPKG-hSOS expression vector into competent cdc25H cells, whichwere then grown for 4 days at 25 C on minimal glucose plates. Colonieswere isolated and tested for galactose-dependent growth at 36 C. Plas-mids were extracted from three initially positive colonies, and theinserts were sequenced before retransformation in cdc25H cells to-gether with pPKG-hSOS. Conventional yeast transformation and ma-nipulation protocols were used. Cells were replica-plated onto eitherglucose- or galactose-minimal medium containing relevant amino acids,according to the manufacturers protocols.

ImmunocytochemistryHEK-NPRA and HEK293 control cellswere incubated in 4-well chamber slides at a density of 100,000cells/cm2 in Dulbeccos modified Eagles medium with 10% fetal bo-vine serum and 1% penicillin/streptomycin solution (Invitrogen). Af-ter fixation in 3.7% formaldehyde, cells were permeabilized with 0.3%Triton X-100 in phosphate-buffered saline for 10 min and blockedwith 1% horse serum, 0.2% bovine serum albumin in phosphate-buffered saline. Fixed slides were incubated with a primary antibodymixture containing 0.1 g/ml rabbit anti-PKG I (Stressgen) or rabbitanti-PKA (Santa Cruz Biotechnology) and 0.7 g/ml mouse anti-FLAG M2 (Sigma). The immunogen used to generate the anti-PKG Iantisera has no homology to PKG II making cross-reactivity ex-tremely unlikely. Cells were blocked for 1 h and then for an additionalhour with fluorescein isothiocyanate or Cy5-conjugated donkey sec-ondary antibodies (Jackson Laboratories); cells were then mountedon coverslips using Gel/Mount mounting medium (Biomedia Corp.).Cardiac cells were treated identically, except fixed cells were incu-bated with a 1:1000 dilution of rhodamine-phalloidin (MolecularProbes, Eugene, OR) for 1 h. Fluorescent imaging was performed witha Leica DMRA upright fluorescent microscope, and images wereacquired with a Hamamatsu ORCA2 CCD camera.

Cardiac Cell CultureThe care of all animals used in this researchwas in accordance with institutional guidelines. Ventricular cardiaccells from 12-day-old Harlan Sprague-Dawley rats were prepared asdescribed previously (33). Ventricles were dissected free from atria andquartered. Myocytes were dissociated in trypsin and DNase I andpreplated to remove non-myocyte cells. In all experiments, cells wereplated on gelatin-coated chamber slides (400,000 cells/chamber) andmaintained overnight at 5% CO2 in Dulbeccos modified Eagles mediumwith 17% Media 199, 10% horse serum, and 5% fetal bovine serum.Cells were incubated in 80% Dulbeccos modified Eagles medium, 20%Media 199 (Invitrogen), and phenylephrine (1 M) 72 h beforetreatments.

Preparation of Plasma MembranesPlasma membranes were madeas desc