Molecular Cloning of the Rat Adipocyte Hormone …THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 268, No. 25, hue of September 5, pp. 18573-18579 1993 Printed in I(I.s.A. Molecular Cloning

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 268, No. 25, h u e of September 5, pp. 18573-18579 1993 Printed in I(I.s.A.

Molecular Cloning of the Rat Adipocyte Hormone-sensitive Cyclic GMP-inhibited Cyclic Nucleotide Phosphodiesterase*

(Received for publication, December 17, 1992, and in revised form, May 3, 1993)

Masato TairaSQT, Steven C. HockmanQ, Juan C. Calvo(1, Masanori Taira**$$, Per BelfrageQQ, and Vincent C. ManganielloQ From the $Laboratory of CeUular Metabolism, National Heart, Lung, and Blood Institute, (1 Bone Research Branch, National Institute of Dental Research and **Laboratory of Molecular Genetics, National Institute of ChiM Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, lsecond Department of Internal Medicine and $$the Department of Biochemistry, Chiba University School of Medicine, Chiba 280, Japan, and the $§Department of Physiological Chemistry, University of Lund School of Medicine, P.O. Box 94, 5-221 00 Lund, Sweden

Two distinct but related cGMP-inhibited cyclic nucleotide phosphodiesterase (cGI PDE) cDNAs were cloned from rat adipose tissue cDNA libraries. The open reading frame (3324 base pairs) of RcGIPl encodes 1108 amino acids, including a hydrophobic membrane-association domain in the NHa-terminal portion and, in the COOH-terminal portion, a putative catalytic domain conserved among all mammalian PDEs which is preceded by a putative regulatory domain that contains three consensus CAMP-dependent protein kinase phosphorylation sites and followed by a hydrophilic COOH-terminal domain. The carbosyl-terminal portion including the conserved domain was expressed as a glutathione S-transferase fusion protein and es- hibited CAMP PDE activity which was inhibited by cilostamide, a specific cG1 PDE inhibitor. RcGIP1 cDNA hybridizes strongly with RNA from isolated adipocytes, and its mRNA increases dramatically during differentiation of 3T3-Ll adipocytes.

The deduced sequence of the second partial cDNA clone (RcGIP2 clone 63B) is highly homologous to the corresponding region of human cardiac cGI PDE cDNA. RcGIP2 cDNA hybridized strongly with rat cardiac tissue RNA and weakly if at all with RNA from rat adipocytes or 3T3-Ll fibroblasts or adipocytes. We suggest that RcGIPl represents the hormone-sensitive, membrane-associated rat adipocyte cGI PDE and RcGIP2, a cGI PDE from vascular elements in rat adipose tissue.

CAMP is an important intracellular second messenger medi- ating the hormonal regulation of triacylglycerol hydrolysis in adipose tissue (1, 2). Lipolytic hormones, such as catecholamines, increase cAMP content and activate the hormone- sensitive lipase via activation of CAMP-dependent protein kinase (protein kinase A) (1). Protein kinase A also phospho- rylates and activates the adipocyte hormone-sensitive cGMP-

* This investigation waa supported in part by the Mochida Me- morial Foundation for Medical and Pharmaceutical Research, Swed- ish Medical Research Council Grant 3362, the Nordic Insulin Foun- dation, and the A. PHhlsson’s Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) 22867.

3 TO whom correspondence shouldbe addressed: Rm. 5N-307, Bldg. 10, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496- 5194; Fax: 301-402-1610.

inhibited cyclic nucleotide phosphodiesterase (cGI PDE),’ presumably related to “feedback” regulation of cAMP concen- trations (3-9). Insulin decreases cAMP and exerts its antili- polytic action at least in part via phosphorylation and activation of the same hormone-sensitive cGI PDE (8-12), which has been purified from microsomal fractions of rat adipose tissue (13). Activation by insulin and catecholamines is associated with phosphorylation of the adipocyte cGI PDE at perhaps distinct serine sites by insulin-dependent serine kinase(s) and protein kinase A, respectively (8, 9). In the presence of insulin and isoproterenol, there is concomitant phosphorylation and activation of the cGI PDE, which is associated with insulin-induced reduction in hormone-stim- ulated protein kinase A and lipolysis (8-10). To define at the molecular level the signaling pathways and mechanisms involved in regulation of the adipocyte cGI PDE by insulin and catecholamines and its role in lipolysis, we have isolated the cDNA for the adipocyte cGI P D E its mRNA is expressed predominantly in adipocytes and is dramatically induced during differentiation of 3T3-Ll adipocytes. We have isolated also a second partial cGI PDE cDNA from adipose tissue which is highly homologous to a cGI PDE cDNA recently cloned from a human cardiac cDNA library (14). The mRNA of this cGI PDE is not apparently expressed in rat or 3T3-Ll adipocytes. These and other observations reported here support the idea that these two cGI PDE isoforms are products of different genes and infer that other cGI PDE isoforms may exist.

MATERIALS AND METHODS Cloning of cGI PDE cDNA-From amino acid sequences of several

overlapping peptides of a purified human platelet cGI PDE (14); a peptide sequence was selected: YNVTDDKYG(R or N)LTGNIPAL ELMALYVAAAMHDYDHPGRT. Four degenerate oligonucleotides (a-d, based on the undZiGiTZquences) were synthesized on an Applied Biosystems 380B DNA synthesizer: sense primer (a) CCGG ATCCTA(CT)AA(CT)GT(AGCT)AC(AGCT)GA(CT)GA(CT)AA(A G)TA, corresponding to YNVTDDKY plus a BamHI restriction site; a degenerate probe (b) GA(GCT)GCTGT(GCT)TTCAC(AGT)GAC

GGTCGACCAT(AGCT)GC(AGCT)GC(AGCT)GC(AGCT)AC(AG) (CT)T(AG)G corresponding to TGNIPAL; antisense primers (c) G

The abbreviations used are: cGI PDE, cGMP-inhibited cyclic nucleotide phosphodiesterase; HCAR, human cardiac; PCR, polymerase chain reaction; SD, Sprague-Dawley; GST, glutathione S-transferase; IPTG, isopropyl-8-D-thiogalactopyranoside; IBMX, methylisobutylxanthine; DEX, dexamethasone; RACE, rapid amplification of cDNA ends; PAGE, polyacrlyamide gel electrophoresis; CAPS, 3- (cyclohexy1amino)propanesulfonic acid; bp, base pair; kb, kilobase.

*E. Degerman, M. Moos, Jr., A. Rasch, V. Vasta, E. Meacci, C. Smith, S. Lindgren, K.-E. Anderson, P. Belfrage, and V. Mangan- iello, manuscript in preparation.

18573

18574 Molecular Cloning of Rat Adipocyte cGI PDE TA corresponding to WAAAM plus a SalI site, and (d) GGGTCGA C(AGCT)C(GT)(AGCT)CC(AGCT)GG(AG)TG(AG)TC(AG)TA corresponding to DHPGRT plus a Sari site. The first round of polymerase chain reaction (PCR) amplification was carried out in a total volume of 100 pl containing 50 mM KCl, 10 mM Tris-HC1 (pH 8.3), 1.5 mM MgClZ,O.Ol% gelatin, 200 p~ dNTPs, and 1 unit of Taq DNA polymerase (Perkin-Elmer Cetus), with 50 ng of X phage DNA of a commercially available X ZAP I1 human cardiac cDNA library (Stratagene) as a template and 100 p~ of primers (a) and (d), under conditions of denaturation at 94 "C for 1 min, annealing a t 37 "C for 1 min for the first two cycles, and at 50 "C for 1 min for the next 28 cycles and extension at 72 "C for 1 min. We initially used a human cardiac cDNA library because a human megakaryocyte library was not available and antibodies raised against the human platelet cGI PDE cross-reacted with and immunoprecipitated human sarcoplasmic reticulum cGI PDE (14). The second round of PCR was done with 1 pl of the first PCR reaction mixture as a template and 100 pM primers (a) and (c) under the same conditions except that annealing was carried out a t 50 "C. PCR products were separated in 2% agarose gel, transferred to nitrocellulose filter, and hybridized with 3ZP-labeled probe (b) labeled with T4 polynucleotide kinase and [-p3'P]ATP. The 130-bp fragment, which hybridized with the probe (b), was inserted into Bluescript plasmid, isolated by colony hybridization, and sequenced by dideoxynucleotide methods described earlier (14).

The 130-bp fragment, labeled with [a-32P]dATP by using a random primer kit (Stratagene), was used to screen 2 X lo6 plaques of a rat adipose tissue cDNA library (supplied by Dr. A. Kimmel, NIDDK, NIH) by plaque hybridization. Hybridization was carried out in 0.5 M sodium phosphate buffer (pH 7.0), 1 mM EDTA, 1% bovine serum albumin, 7% SDS at 60 "C overnight (15). Replicate filters were washed in 6 X SSPE (1 X SSPE = 0.1 M sodium phosphate (pH 7.0), 0.15 M NaCl, 0.6 mM EDTA) and 0.1% SDS a t room temperature and exposed to Kodak XAR film with intensifying screen at -70 "C overnight. Eight independent clones were isolated and plaque-puri- tied. PCR amplification of the 130-bp fragment from the cDNA of the eight clones with degenerate sense primer (a) and antisense primer (d) suggested the presence of two distinct but related cDNAs (designated as RcGIPl and RcGIP2), which was confirmed by sequencing several of the cDNA clones by dideoxynucleotide methods (14). One partial RcGIPl cDNA clone 513 (see Fig. L4) was then used to screen rat adipose tissue cDNA library 11.

Construction and Screening of Rat Adipose Tissue cDNA Library IZ-Total RNA was isolated from epididymal adipose tissue of Spra- gue-Dawley rats by the acid guanidinium cyanate-phenol-chloroform extraction procedure (16) and used to construct a cDNA library (cDNA synthesis kit, Pharmacia LKB Biotechnology Inc.) in X ZAP I1 (according to the manufacturer's protocol). Briefly, the first cDNA strand was synthesized with olig(dT) primer and Maloney virus reverse transcriptase. After treatment with RNase H, the second strand was synthesized with polymerase I (17) and EcoRIINotI adap- tors ligated to blunt-ended cDNA. The double-stranded cDNA was then ligated into EcoRI sites in X ZAP 11. Approximately 2 X lo6 plaques were screened with the partial RcGIPl cDNA clone 513 (bases 1919-2634), labeled with [ c ~ - ~ ' P ] ~ A T P (random primer kit, Stratagene). Replicate filters were incubated in prehybridization medium at 65 "C overnight, hybridized at 65 "C overnight as described above, washed at 65 "C in 0.5 X SSC, 0.1% SDS, and exposed to x- ray film. Five independent overlapping clones were isolated, plaque- purified, and used for sequencing (Fig. LA).

RACE Procedure to Determine 5' Region of Adipocyte cGI PDE cDNA-As described by Frohman and Martin (18), first strand cDNA was synthesized at 70 "C with 10 units of thermostable rTth reverse transcriptase (Perkin-Elmer Cetus), cDNA-specific internal primers, and 0.5 pg of total RNA extracted from epididymal adipose tissue of SD rats (16), and, after purification with Centricon 100, tailed with dATP using 10 units of terminal deoxynucleotidyltransferase (Life Technologies, Inc.). dTI7-adapter primer, 5"CGGGATCCCGGACA TCGATTTTTTTTTTTTTTTTT-3', and adapter primer, 5'-CGG GATCCCGGACATCGA-3', were synthesized. The single-stranded cDNA was amplified with 1 p~ internal cDNA-specific primer, dT17- adapter primer, and adapter primer (ratio 1:2) in the buffer described above. The PCR was carried out for 30 cycles of 94 "C for 1 min, 62 "C for 1 min, and 72 "C for 1 min. RACE was performed seven times with different internal primers, and 79 independent clones were identified by colony hybridization using internal probes (Fig. L4). Sequences from several independent clones were determined.

Production of Fusion Proteins-Portions of RcGIPl cDNA (bases 1856-3396; amino acids 598-1108), and RcGIP2 clone 53B (1053 bp)

A RAT ADIPOCYTE cGI PDE (RcGIPl)

H (RACE) -I-

CONSERVED CATALYTIC DOMAIN

I I I

MEMBRANE-ASSOCIATION ADDITIONAL REGION DOMAIN

B

DISTRIBUTION OF mRNA FOR RcGIP1 AND RcGIP2 cGI PDE IN RAT TISSUES

RcGlPl RcG I P2

28s - 18s

8.1 - 6.5 -

- 5.3 5.1 - ( 4

" '

6 28s

4- 18s

F H B L F H B L

to nt 1919-2634 in RcGIP1) was isolated from a rat epididymal adipose FIG. 1. A, Sequencing strategy for RcGIP1. Clone 513 (corresponding

tissue cDNA library supplied by Dr. A. Kimmel, NIDDK, NIH, by plaque

cardiac cDNA library (Stratagene) using degenerate oligonucleotide hybridization with a 130-bp fragment amplified by PCR from a human

primers based on peptide sequences derived from a purified human platelet cGI PDE. Clone 513 was then used in screening a second adipose tissue cDNA library. Five additional independent overlapping clones (three clones (331 (nt 144-3248), 271 (nt 1369-4381), and 431 (nt 716-4264)) are presented) were isolated and sequenced as indicated to determine 4231 bp of RcGIP1. By the RACE procedure, 79 independent clones were

additional 150 bp of 5' sequences. B, Distribution of RcGIPl and RcGIP2 isolated, several of which were sequenced and used to determine an

by electrophoresis in 1% agarose-formaldehyde gel, transferred to nitro- in rat tissues. Total RNA (10 pg) from the indicated tissues was separated

cellulose filters, which, after prehybridization, were hybridized with 32P- labeled RcGIPl clone 513 ( A ) or RcGIP2 53B (Fig. 2B) (random priming kit, Stratagene) at 68 "C overnight. Filters were washed several times with 6 X SSPE at 68 'C (total time, 2 h) and exposed to Kodak x-ray film at -70 "C for 7 days. mRNA size was estimated using ribosomal RNA (18 S, 1.9 kb and 28 S, 4.5 kb) as standards. C, nucleotide sequence and deduced amino acid sequence of RcGIPl determined by RACE and from overlapping cDNA clones in Fig. IA.

Molecular Cloning of Rat Adipocyte cGI PDE 18575

18576 Molecular Cloning of Rat Adipocyte cGI PDE

4 5.3

aa.1

*6.5

FIG. 2. Northern blots hybridized with probes to detect (A) RcGIPl mRNA or ( B ) RcGIP2 mRNA. Total RNA (10 pg/lane) was isolated from the indicated cells and tissues, subjected to electrophoresis in a 1% agarose-formaldehyde gel, and transferred to a nylon membrane as described under “Materials and Methods.” Blots were prehybridized at 60 “C and hybridized at 60 “C overnight with 32P- labeled DNA probes (the RcGIPl probe, clone 431, nt 716-4264, or RcGIP2, clone 53B) as described under “Materials and Methods.” Following a room temperature wash, blots were washed (three times) for 20 min at 60 “C with 0.5 X SSC and 0.1% SDS. Films were exposed for 3 days at -70 “C with an intensifying screen. Numbers on the right indicate size of RNA (kb). Lane 1, rat adipocyte; lane 2, rat adipose tissue; lane 3, rat heart; lane 4, 3T3-Ll fibroblast; lane 5, 3T3-Ll differentiated adipocyte.

were amplified by PCR with specific primer sets: 5”CCGGATCCAC GATTGAACATTGTGGA-3’ and 5’-CCGGATCCTCTTCTCATT CAAACATTTG-3’ corresponding, respectively, to bases 1856-1875 and 3377-3396 in RcGIP1, and 5’-CGTGGATCCACGGCCGATTC CTGGCCTC-3’ and 5’-CAATTCAAGCTTCTTGATAGCCTGGA TTTG-3’ corresponding, respectively, to bases 1-18 and 1036-1053, in RcGIP2. (Sequences underlined are restriction enzyme linkers for BarnHI and HindIII.) PCR products were digested with appropriate restriction enzymes, inserted into the corresponding cloning sites of pGEXl vector, and used to transform DH5a competent cells (Life Technologies, Inc.). Transformed cells were streaked on LB/ampicillin (50 pglml) plates. After colony hybridization, single positive colonies were picked and grown in LB/ampicillin medium. Trans- formed Escherichia coli (ODm = --0.5) were amplified and then incubated with or without 0.5 mM isopropyl-8-D-thiogalactopyranoside for 2 h a t 37 “C. Cells were harvested, suspended in 1 X phosphate-buffered saline, sonicated, and centrifuged (14,000 X g, 1 min). Portions of the supernatants containing RcGIPl and RcGIP2 glutathione S-transferase fusion proteins were incubated with glutathione- agarose beads (Pharmacia) for 30 min at 4 “C. Fusion proteins were eluted with 50 mM reduced glutathione in 50 mM Tris-HC1 buffer, pH 8.0. Cell lysates (10 pl) or fusion protein (15 pl) were mixed with 125 mM Tris, 10% glycerol, 1% SDS, 10% 2-mercaptoethanol, 0.05% bromphenol blue and subjected to SDS-PAGE (10% gels, 60 min, 100 V). Proteins were stained with Coomassie Blue (one gel), and transferred (from a second gel) to poly(viny1idene fluoride) membrane (45 min, 50 V, 0 “C) in 10 mM CAPS, pH 10, 10% methanol. After blotting, the poly(viny1idene fluoride) membrane was incubated 1 h in blocking solution (0.2% I-Block reagent (Tropix) and 0.3% Tween 20 in phosphate-buffered saline), and incubated for 1 h at room temperature with anti-human platelet or bovine adipose tissue cGI PDE IgG purified by chromatography of antisera on protein A. The membranes were then washed three times with blocking solution. Immunoreactivity was detected with anti-rabbit IgG-linked horse radish peroxidase by the Enhanced Chemiluminescent Method (Amersham Corp.).

Northern Blots-Total RNA was extracted from various tissues and cultured cells by the method of Chomczynski and Sacchi (16). RNA quality was assessed by electrophoresis of -10 pg of total RNA on a 1% agarose/formaldehyde gel; the 28 S and 18 S ribosomal RNA bands exhibited an approximate 2/1 ratio upon staining with ethidium bromide, indicating minimal degradation of the preparations.

For Northern blots, approximately 10 pg of total RNA were subjected to electrophoresis in 1% agarose/formaldehyde gels (20 V, 20

h). Gels were stained with ethidium bromide and photographed, and RNA was then transferred to nylon membranes (Schleicher & Schuell) and fixed using a UV cross-linker (Stratalinker, Stratagene).

Differentiation of 3T3-Ll Adipocytes-3T3-Ll fibroblasts were grown in six-well plates (35 mm) in 2 ml of Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. Two days after cells were confluent, fresh medium containing 0.5 mM methylisobutylxanthine (IBMX) and 1 p~ dexamethasone (DEX) were added; 2 days later, 1 p~ insulin was added. After 2 days, DEX and IBMX were removed, and fresh medium containing insulin was added. Every 2 days thereafter, medium was replaced with fresh medium without insulin, IBMX, or DEX. Fully differentiated 3T3-Ll adipocytes were harvested 1 week after addition of DEX and IBMX. Total RNA from undifferentiated 3T3-Ll fibroblasts and differentiated adipocytes was prepared as described above (16).

RESULTS AND DISCUSSION

An overlapping peptide sequence of 38 amino acids was obtained from purified human platelet cGI PDE (14): Be- cause this peptide sequence is similar to part of the putative catalytic domain conserved among all known mammalian PDEs (19), oligonucleotides based on the platelet cGI PDE sequence were synthesized and used to amplify a 130-base pair (bp) cDNA fragment from a human cardiac cDNA library by PCR. The deduced amino acid sequence of the 130-bp fragment corresponded to the platelet cGI PDE sequence. The cDNA fragment was then used as a probe to screen a rat adipose tissue cDNA library under low stringency conditions. Eight independent clones were isolated, which contained two distinct but related partial cGI PDE (designated as RcGIPl and RcGIP2) cDNAs. One partial RcGIPl cDNA clone (513) (Fig. LA) hybridized predominantly with an -5.3-kb mRNA in adipose tissue RNA (Fig. 1B), whereas RcGIP2 cDNA (clone 53B) hybridized more strongly with rat cardiac tissue RNA than with adipose tissue RNA; neither hybridized strongly with RNA from rat brain or liver (Fig. 1B).

Since RcGIPl cDNA clone 513 hybridized with mRNA predominantly expressed in rat adipose tissue, it was used to screen a second rat adipose tissue cDNA library. Five independent overlapping cDNA clones were isolated and used for sequencing; the 5“terminal portion was determined by the RACE procedure (Fig. LA). Based on the overlapping clones, RcGIPl cDNA is 4381 bp in length (Fig. IC). The sequence has an open reading frame encoding 1108 amino acids (Fig. IC), starting with a methionine codon at nt 65, and ending with a termination codon at nt 3390, followed by 1 kb of 3‘- untranslated region which does not contain a poly(A) tail (Fig. IC). Although the methionine codon conforms to Ko- zak’s rules ( G G G C T a A ) (20) translation could initiate further upstream, because the cDNA sequence 5’ to base 65 does not contain an in-frame termination codon.

On Northern blots (Fig. 2 4 1 , RcGIPl cDNA clone 431 (nt 716-4264) (cf. Fig. IA) hybridized much more strongly with RNA extracted from isolated adipocytes (lane 1 ) than with that from adipose tissue (lane 2), consistent with the view that RcGIPl is highly, and perhaps specifically, expressed in adipocytes. On the other hand, RcGIP2 cDNA (clone 53B) hybridized primarily with RNA from cardiac tissue (lane 3), and not with that from adipocytes (lane 1 in Fig. 2B). Fur- thermore, RcGIPl mRNA expression correlated with differentiation of 3T3-Ll adipocytes (lane 5 in Fig. 2A), whereas RcGIP2 mRNA was not detected in either undifferentiated 3T3-Ll fibroblasts or differentiated 3T3-Ll adipocytes (Fig. 2B). Since hormone-sensitive cGI PDE activity is induced during differentiation of 3T3-Ll cells (21), these data support the idea that RcGIPl cDNA encodes the hormone-sensitive adipocyte cGI PDE. Since cGI PDE mRNA is apparently induced by dexamethasone and methylisobutylxanthine alone

Molecular Cloning of Rat Adipocyte cGI PDE 1 a m A

HCAR . .

I , . . , I

RcGIPZ(partia1 clone, 5 3 B ) ’ m ” t

B MRKDERERDT PAMRSPPPPP PPATATMSP PESLRNGYVK SCVSPLRQDP 50

PRSFFFHLCR FCNVEPPAAS GARLSLA ALAAFVLAAL LGAGPERWAA

150 AATGLRTLLS ACSLSLSPLF SIACAFFFLT CFLTRAQRGP DRGAGSWWLL

100

ALPACCYLGD FMWQWWSWL RGEPAAAAAG RLCLVLSCVG LLTLAPRVRL 200

RHGVLVLLFA GLVWWVSFSG LGALPPALRP LLSCLVGGAG CLLALGLDHF 250 I

GASPPP RSASTADEKV PVIRPFWRR CVSLGESAAG YYGSGKMFRR 300

ELPCISREQ MILWOWDLKQ WCKPHYQNSG GGNGVDLSVL NEARNMVSDL 350

LIDPSLPPQV ISSLRSISSL MGAFSGSCRP KINSFTPFPG FYPCSEVEDP 400

VEKGDRKLHK GLSSKPSFPT A Q L R R G A S GLLTSEHHSR WDRSGGKRPY 450

QELSVSSHGC HLNGPFSSNL MTIPKQRSSS VSLTHHAGLR RAGALPSPSL 500

LNSSSHVPVS AGCLTNRSPV GFLDTSDFLT KPSVTLHRSL GSVSSMDFH 550

QYLRNSDSSL CSSCGHQILK YVSTCEPDGT DHHNEKSGEE DSTVFSKERL 600

NIVETQEEET VKEDCRELFL EGDDHLMEEA QQPNIDQEVL LDPMLVEDYD 650

SLIEKHSNWN FQIFELVEKH GEKSGRILSQ

EFMNYFRALE NGYRDIPYHN RVHATDVLHA

TETKADSDAR LSSGQIAYLS SKSCCIPDKS

AMHDYDHPGR TNAFLVATNA PQAVLYNDRS

NFLLNLDHME FXRFRFLVIE AILATDLKKH

WSSENDRLLV CQVCIKLADI NGPAKDRDLH

LGLPISPFMD RSSPQLAKLQ ESFITHIVGP

GDDTESDDDD DDDDDDDDDD DEELDSDDEE TEDNLNPKPQ RRKGRRRIFC 1050

Q W H L T E N H KIWKEIIEEE EKCKAEGNKL QVDNASLPQA DEIQVIEEAD 1100

EEEEQMFE 110s

C 1

0 RcGlPl

-50 1 500 1000

0 HCAR

-50 I 500 1000

FIG. 3. A, structure of RcGIP1, human cardiac cGI PDE (HCAR), and partial RcGIP2 clone 53B. Black box, membrane association domain; dark dotted box, catalytic domain; light dotted box, insert of 44 amino acids. Clone 53B encodes partial RcGIP2 protein. Numbers are percentage amino acid identity between these clones in the indicated segments. RcGIPl and RcGIP2 GST fusion proteins were expressed in E. coli as described under “Materials and Methods”; clone n.2 is an independent partial HCAR cDNA clone which encodes

TABLE I CAMP phosphodiesterase activities of RcGIPl -GST fwwn protein

expressed in E. coli Whole lysates of E. coli transformed with pGEX-RcGIP1-1 (GST-

RcGIP1) or pGEX-RcGIP1-1R (control) were prepared as described under “Materials and Methods.” pGEX-RcGIP1-1 encodes a GST fusion protein of RcGIPl carboxyl-terminal region (as shown in Fig. 3, A and B ) containing the entire conserved region. pGEX-RcGIP1- 1R has the same insert as pGEX-RcGIP1-1 but in the reverse orientation. The PDE activities of lysates were assayed with or without 1.6 ~ L M cilostamide as described (9). Numerical figures show mean f S.D. of six or four determinations as indicated in parentheses.

Clone Enzyme activity

None Cilostamide

pmol/min/mg protein GST-RcGIP1 9.38 2 2.77 0.597 f 1.00 (6) Control 0.883 2 0.271 0.657 f 0.217 (4)

during the early phases of 3T3-Ll adipocyte differentiation (data not shown), study of the regulation of expression of the adipocyte cGI PDE gene may provide additional insights into the mechanisms of adipocyte differentiation.

As seen in Fig. 3A, the nucleotide sequence and deduced amino acid sequence of the partial RcGIP2 cDNA clone 53B is very similar to the corresponding region of the HCAR cGI PDE cDNA recently cloned from a human cardiac tissue cDNA library (14). The 1116-bp open reading frame of the partial RcGIP2 cDNA clone 53B (data not shown) (Fig. 3A) encodes 372 amino acids which closely correspond t o the carboxyl-terminal region of HCAR cGI PDE (14). Identity of clone 53B and the corresponding region of HCAR is 88% at the amino acid level and 85% at the nucleotide level, whereas the identity of clone 53B and the corresponding region of RcGIPl is 63% and 61%, respectively (data not shown). These results, which indicate that HCAR and RcGIP2 are more closely related than RcGIPl and RcGIP2, suggest that there may be considerable conservation of certain cGI PDE isoforms during evolution. RcGIP2 most likely represents a cGI PDE derived from vascular elements in adipose tissue, rather than from adipocytes. In this regard, it has been recently reported that adipocytes contribute only about one-third of total adipose tissue cAMP PDE activity (assayed at 1 phi cAMP substrate) (22), and cGI PDE activity has been dem- onstrated in extracts from rat ventricular myocytes (23) and rat aorta (24). Since the nucleotide differences between RcGIPl and RcGIP2 are such as to rule out their generation via alternative splicing mechanisms, it appears that the two cGI PDE cDNAs isolated from the rat adipose tissue libraries encode two cGI PDE isoforms that are products of distinct genes. Neither RcGIPl or RcGIP2 cDNAs strongly hybridized with total RNA from rat liver (a tissue known to express hormone-sensitive cGI PDE activity (25, 26)). Whether this

amino acids 613-1108 of HCAR (14); These three cDNAs were inserted into pGEX-1 and expressed as p fusion proteins in E. coli (cf. below). B, deduced amino acid sequence of the rat adipocyte hormone-sensitive cGI PDE encoded by RcGIPl cDNA. Upper box indicates the hydrophobic membrane association domain; lower box encloses the conserved catalytic domain with the 44-amino acid insertion underlined (in other cGI PDEs, we also find insertions of 44 amino acids, which do not align within the conserved domains of other PDE families). Three putative protein kinase A phosphorylation sites (RRXS), which are located in the putative regulatory domain between the two boxes, are also underlined. C, hydropathy index. Averaged hydropathy index values for putative RcGIPl and HCAR proteins (14) were calculated by the program SOAP (PC GENE, Intelligenetics, Inc.) with a window size of 15 amino acids. Amino acid number is on the horizontal axis.

18578 Molecular Cloning of Rat Adipocyte cGI PDE

A M 1 2 3 4 5 6 7

M 1 2 3 4 5 6 7

M 1 2 3 4 5 6 7 -7" -II

- -I -1

-1 FIG. 4. Expreseion of RcGIPl and RcGIP2 as GST fusion

proteins in E. coli. RcGIPl fusion protein includes GST plus 511 amino acids (amino acids 598-1108) of the carboxyl-terminal region of RcGIPl protein (pGEX-RcGIP1-1); RcGIP2 fusion protein, GST plus 351 amino acids (clone 53B, Fig. 1B) of RcGIP2 protein (pGEX- RcGIP2-1). Cell lysates and purified fusion proteins were subjected to SDS-PAGE (10% gel). Open triangles indicate RcGIPl fusion protein and closed triangles indicate RcGIP2 fusion protein. Western blot ( A ) with anti-bovine adipose tissue cGI PDE antibody and ( B ) with anti-human platelet cGI PDE antibody. The enhanced chemi- luminescence detection method (ECL, Amersham Corp.) was used (30). The antisera and second antibody also reacted with bacterial proteins, which are seen in lanes 2 through 6. C, Coomassie Blue staining. Lane M , molecular size markers from top to bottom: 200, 97.4, 68, 43, 29, and 18.4 kDa indicated by lines at the left. Lane 1, purified GST protein alone; lane 2, GST-RcGIP1 cell lysate (uninduced); lane 3, GST-RcGIP1 cell lysate (induced by isopropyl-8-D- thiogalactopyranoside; lane 4, purified GST-RcGIP1 (induced); lane 5, GST-RcGIP2 cell lysate (uninduced); lane 6, GST- RcGIP2 cell lysate (induced); lane 7, purified GST-FkGIP2 (induced).

reflects the existence of another cGI PDE isoform(s) is the subject of current studies.

Portions of RcGIPl (amino acids 598-1108) (Fig. 3, A and B ) and RcGIP2 (clone 53B) (Fig. 3A) cDNAs were expressed as glutathione S-transferase fusion proteins in E. coli. In RcGIP1, amino acids 696-980 represent the putative catalytic domain conserved among all mammalian PDEs (19,27) (Fig. 3, A and B). RcGIP2 clone 53B is very similar to the corresponding region in HCAR (Fig. 3A) and to HCAR clone n.2 which encodes amino acids 613-1108 in the carboxyl- terminal region of HCAR cGI PDE (14) (Fig. 3A). HCAR clone n.2 (amino acids 613-1108) (14) and the RcGIPl fusion protein (amino acids 598-1108) include the entire conserved domains of RcGIPl (amino acids 696-980) and HCAR (amino acids 729-1011), plus some additional NHz- and COOH-terminal amino acids. RcGIP2 clone 53B, however, does not encode the entire conserved domain (Fig. 3A). As shown in Table I,

the RcGIPl fusion protein exhibited cAMP PDE activity that was inhibited by cilostamide, indicating that RcGIPl belongs to the cGI PDE gene family (25). In addition, the RcGIPl fusion protein reacted more strongly than did RcGIP2 with the anti-bovine adipose tissue cGI PDE antibody (Fig. 4) (compare the relative amounts in panel C and the immunoreactivity in panek; A and B of these proteins; i.e. compare the RcGIPl band (open triangle) in lane 4 to the RcGIP2 band (closed triangle) in lane 7 in each panel). This antibody has been used for immunoprecipitation and immunoblotting of hormone-sensitive cGI PDE in rat adipocytes (8,9).

In contrast, anti-human platelet cGI PDE antibody (14), which immunoprecipitates human cardiac sarcoplasmic reticulum cGI PDE, reacted with RcGIP2 fusion protein more strongly than with RcGIPl fusion protein (Fig. 4B). HCAR clone n.2 has been expressed as a GST fusion protein in E. coli and exhibited cAMP PDE activity inhibited by OPC 3911 and cGMP, but not rolipram (14). The RcGIP2 fusion protein encoded by clone 53B did not exhibit detectable cGI PDE activity (data not shown). Since RcGIP2 clone 53B is highly homologous to HCAR clone n.2, but lacks codons for 44 amino acids in the conserved domain and -100 amino acids NH2- terminal to the conserved domain, these results indicate that the entire conserved domain (and perhaps some additional sequences) in cGI PDEs is required for catalytic activity. In fact, a recent report of the mapping of the catalytic domain of a recombinant rolipram-sensitive CAMP-specific PDE suggested that the catalytic domain includes the conserved domain as well as a region of about 68 amino acids NHz-terminal to it (28).

The molecular weight calculated from the deduced amino acid sequence of RcGIPl (Fig. 3B) is 123,091. The subunit molecular mass of the phosphorylated hormone-sensitive cGI PDE in rat adipocytes was estimated at -135 kDa by SDS- PAGE (8,9). The PDE mass may be overestimated by SDS- PAGE, perhaps due to the presence of highly acidic residues in the carboxyl-terminal region. The amino acid sequence encoded by RcGIPl cDNA predicts the presence of at least four major domains. Although one region (amino acids 696- 980, Fig. 3, A and B) is clearly related to the putative catalytic domain common to other PDEs (14, 19, 27), in this domain of cGI PDE an insertion of 44 amino acids (amino acids 740- 783), which does not align with other PDE sequences, was identified (Fig. 3, A and B). An insertion of 44 different amino acids is present also in RcGIP2 (Fig. 3A) and HCAR (14), from which we infer that this region may be both specific to the cGI PDE gene family and important in identification of individual members of the family. The deduced amino acid sequences of adipocyte cGI PDE (RcGIP1) and human cardiac tissue cGI PDE (HCAR) exhibit very similar hydropathy index patterns (Fig. 3C) despite quite limited identity of the sequences except in the catalytic domain (Fig. 3A), and it appears that these two cGI PDEs might possess similar three dimensional structural and functional domains. Putative membrane association domains (amino acids 73-251) in RcGIP1, located in the amino terminal region (Figs. 3, A, B, and C ) , are of interest since the hormone-sensitive adipocyte cGI PDE as isolated is associated with microsomes (25, 29). A hydrophilic region (amino acids 994-1049) is found in the COOH-terminal domain. Recent evidence also indicates that, out of three possible consensus protein kinase A phosphorylation sites (RRXS) in the putative regulatory domain (amino acids 252-695) (Fig. 3B), one, LRRSSGASGLLTSEHHSR (amino acids 423-440), was very likely phosphorylated on serine 427 in 32P-~GI PDE immunoisolated from solubilized rat adipocyte membranes phosphorylated in vitro with protein

Molecular Cloning of Rat Adipocyte cGI PDE 18579

kinase A.3 Similar approaches should help to identify the site(s) in the adipocyte cGI PDE phosphorylated by the insulin-sensitive intracellular serine kinase(s) (3,8, 9, 12).

The molecular cloning of the adipocyte-specific cG1 PDE should provide new insights into the structure-function rela- tionships of this enzyme and offers a new tool to elucidate in detail molecular mechanisms for the insulin-signaling pathways involved in phosphorylation/activation of the cG1 PDE and inhibition of lipolysis. It is hoped that this information will also facilitate better understanding of obesity and other risk factors important in ischemic heart disease and non- insulin-dependent diabetes mellitus.

Acknowledgments-We thank Drs. M. Vaughan for encourage- ment; T. Oeda for help with RACE methodology; A. Kimmel for supplying a rat adipose tissue cDNA library; E. Degerman and M. Moos for sequencing of peptides from and information concerning human platelet cGI P D E E. Meacci, R. Haun, C. J. Smith, N. Komas, S. Kedev, and L. Wang for advice and assistance with specific procedures; A. Rascon for permission to cite unpublished data; 1. B. Dawid for PCR cloning advice; Dr. T. Kono for review of the manuscript; and C. Kosh for typing the manuscript.

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Molecular Cloning of the Rat Adipocyte Hormone …THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 268, No. 25, hue of September 5, pp. 18573-18579 1993 Printed in I(I.s.A. Molecular Cloning