arden1999, molecular cloning of pancreatic islet-specific glucose-6-phosphatase catalytic subunit...

8/17/2019 Arden1999, Molecular Cloning of Pancreatic Islet-specific Glucose-6-Phosphatase Catalytic Subunit Related Protein

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DIABETES, VOL. 48, MARCH 1999 531

Molecular Cloning of a PancreaticIslet–Specific Glucose-6-Phosphatase CatalyticSubunit–Related ProteinSusan D. Arden, Tobias Zahn, Sabine Steegers, Simone Webb, Barbara Bergman, Richard M. O’Brien,

and John C. Hutton

A pancreatic islet–specific glucose-6-phosphatase–related protein (IGRP) was cloned using a subtractivecDNA expression cloning procedure from mouse insuli-noma tissue. Two alternatively spliced variants that dif-fered by the presence or absence of a 118-bp exon(exon IV) were detected in normal balb/c mice, diabetic

ob / o b mice, and insulinoma tissue. The longer, 1901-bp

full-length cDNA encoded a 355–amino acid protein(molecular weight 40,684) structurally related (50%overall identity) to the liver glucose-6-phosphatase andexhibited similar predicted transmembrane topology,conservation of catalytically important residues, andthe presence of an endoplasmic reticulum retention sig-nal. The shorter transcript encoded two possible openreading frames (ORFs), neither of which possessedHi s1 7 4, a residue thought to be the phosphoryl acceptor(Pan CJ, Lei KJ, Annabi B, Hemrika W, Chou JY: Tr an s-membrane topology of glucose-6-phosphatase. J BiolC h e m 273:6144–6148, 1998). Northern blot and reversetranscription–polymerase chain reaction analysisshowed that the mRNA was highly expressed in pancre-atic islets and expressed more in -cell lines than in an

-cell line. It was notably absent in tissues and cell linesof non-islet neuroendocrine origin, and no other majortissue source of the mRNA was found. During develop-ment, it was expressed in parallel with insulin mRNA.The mRNA was efficiently translated and glycosylated inan in vitro translation/membrane translocation systemand readily transcribed into COS 1, HIT, and CHO cellsusing cytomegalovirus or Rous sarcoma virus promoters.

Whereas the liver glucose-6-phosphatase showed activ-

ity in these transfection systems, the IGRP failed toshow glucose phosphotransferase or phosphatase activ-ity with p-nitrophenol phosphate, inorganic pyrophos-phate, or a range of sugar phosphates hydrolyzed by theliver enzyme. While the metabolic function of theenzyme is not resolved, its remarkable tissue-specificexpression warrants further investigation, as does its

transcriptional regulation in conditions where glucoseresponsiveness of the pancreatic islet is altered.

Diab et e s 48:531–542, 1999

Glucose-6-phosphatase (G-6-Pase) [EC 3.1.3.9]activity is highly expressed in liver and kidney,where it plays a key role in catalyzing the ultimatestep in the gluconeogenic pathway (1,2). The

liver enzyme has been purified (3), cloned (4), and extensivelystudied in both a physiologic context and in the context of the pathology of type 1 glycogen storage disease (5,6). It under-goes reciprocal changes in gene expression with glucokinasein diabetes, consistent with a role in the regulation of hepaticglucose output and consumption (7,8). G-6-Pase in the pres-ence of glucose phosphorylating enzymes forms a “substrateshuttle” or “futile cycle,” which provide exquisite control of metabolic flux through glycolysis (9). The intracellular local-ization of G-6-Pase activity in the endoplasmic reticulum(ER) and outer nuclear envelope (10,11) is suggestive of other roles such as the hydrolysis of sugar phosphatesreleased by trimming of core-glycosylated proteins andactive phosphate transport from the cytoplasm to the ERlumen, which in turn could facilitate Ca 2+ accumulation in thiscompartment by providing a counterion (12). Numerous point mutations (13–15) and splicing abnormalities (16) in the

liver G-6-Pase or the associated transporters (17,18) havebeen documented in association with type 1 glycogen storagedisease in humans and dogs (19). Although variable in phe-notype, in most cases the disease is manifested principally byliver histopathology and fasting hypoglycemia. A pancreaticendocrine phenotype (17) and sudden infant death syndrome(20) have been reported as more rarely observed phenotypicassociations of hepatic G-6-Pase deficienc y.

G-6-Pase activity in islets has been of particular interest inthe context of the potential regulatory importance of the glu-cose substrate shuttle in determining rates of glycolytic fluxand thereby the glucose concentration dependence of secre-tion (21–24). Early studies indicated that glucose-6-phosphate(G-6-P) itself might be an intracellular second messenger in the

From the Department of Clinical Biochemistry (S.D.A.), University of Cam-bridge, Addenbrooke’s Hospital, Cambridge, U.K.; Barbara Davis Center for

Childhood Diabetes (T.Z., S.S., S.W., B.B., J.C.H.), University of ColoradoHealth Sciences Center, Denver, Colorado; and the Department of Molec-ular Physiology and Biophysics (R.M.O.), Vanderbilt University School of Medicine, Nashvi lle, Te n n e s s e e .

Address correspondence and reprint requests to Dr. John C. Hutton, Bar-bara Davis Center for Childhood Diabetes, University of Colorado HealthSciences Center, Box B-140, 4200 E. 9th Ave., Denver, CO 80262. E-mail: j o h n . h u t t o n @ u c h s c . e d u .

Received for publication 23 July 1998 and accepted in revised form 30November 1998.

R.O. is a paid consultant of Oncogene Science.C M V, cytomegalovirus; DMEM, Dulbecco’s modified Eagle’s medium;

ELISA, enzyme-linked immunosorbent assay; ER, endoplasmic reticulum; G-6-P, glucose-6-phosphate; G-6-Pase, glucose-6-phosphatase; IGRP, islet G-6-Pase–related protein; M r , molecular weight; nt, nucleotide; OD, optical density;ORF, open reading frame; PCR, polymerase chain reaction; p-NPP, p-nitrophenol phosphatase; RSV, Rous sarcoma virus; RT, reverse transcription; SSPE, NaCl,Na phosphate, EDTA buffer; UTR, untranslated region.


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stimulus-secretion coupling mechanism. More recently, it hasemerged that G-6-P, along with xyulose 5P, mediates theprocess of gene transcriptional regulation induced by glu-cose or carbohydrate feeding (25). The other key enzymes inthe glucose shuttle, namely glucokinase and the various hex-okinase isoforms, have been extensively characterized(26,27); however, the function and even the very existence of G-6-Pase in islets have remained controversial. Levels of G-6-Pase activity in the islet ranging from zero (28) to up to 10-foldhigher than the hepatic level (29) have been documented.Inhibitor studies indicate that the islet enzyme may be a dif-ferent, or alternatively spliced, gene product from the liver enzyme and exhibit different kinetic characteristics.Increases in islet G-6-Pase in association with fasting hyper-glycemia, increased glucose cycling, and glucose unrespon-siveness have been observed in the ob / ob mouse model of type 2 diabetes (30). Experimental upregulation of islet G-6-Pase using adenoviral vectors encoding the liver enzyme areassociated with reduced glucose responsiveness of insulinsecretion (31). Conversely, mathematical modeling of glucosecycling based on kinetic parameters for the liver enzyme have

discounted a role for the enzyme in regulation of glucosemetabolism and stimulus-secretion coupling in the-cell (32).

We report here the isolation of a series of alternativelyspliced full-length cDNAs from a mouse TC3 insulinoma cDNA library which encode a homolog of the liver G-6-Pase.The mRNA expression is highly specific to the pancreatic islet,indicating a key developmental or regulatory function. Stud-ies of the enzyme expressed in heterologous cells have yet toreveal its catalytic function; however, this may depend on itsintegration into a multimeric membrane complex, which hasnot been reproduced in the simple transfection protocolsused in these experiments. The accompanying article (seepage 543 of this issue) reports the structure and chromoso-mal localization of the gene.

RESEARCH DESIGN AND METHODS

Unless otherwise stated, all molecular cloning procedures were performed by stan-dard protocols (33), and all reagents were of analytical grade and purchased

from Sigma Chemical, Poole, U.K. A previously identified 223-bp insert (nt682–904; Fig. 1) (34) homologous to the liver G-6-Pase open reading frame (ORF)

was radiolabeled by random priming (oligolabeling kit; Pharmacia, Uppsala,

Sweden) with [-32P]dCTP (Amersham, Amersham, U.K.) and used to screen a full-length cDNA library prepared from mouse Rip Tag insulinomas in the plasmid vec-

tor pSVSPORT (35). Sequencing was performed in both directions by the Sanger dideoxy chain termination method on double-stranded templates using synthetic

oligonucleotide primers. In vitro transcription/translation/translocation assays

were performed with rabbit reticulocyte lysate and dog pancreatic microsomalmembranes as previously described (36) using either SP6 polymerase transcribed

pSVSPORT constructs or T7 transcripts from pCDNA3.

The full-length protein was expressed in COS 1 and HIT M2.2.2 cells using a

cytomegalovirus (CMV) promoter construct in pCDNA3 (Invitrogen, Carlsbad, CA)by subcloning a EcoR1 / Xho1 fragment from the original pSVSPORT clones pro-duced in the initial screen. A rat liver G-6-Pase obtained from Rebecca Taub

(University of Pennsylvania School of Medicine, Philadelphia, PA) was cloned into

the same vector using Hi ndIII and XhoI sites. Transfection efficiency was inter-

nally controlled by the cotransfection of -galactosidase in pSVL at an approxi-

mate 1:4 molar ratio. Subconfluent cultures (8.5-cm dishes) were grown in Dul-becco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine

serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. The cells were incu-

bated with a calcium phosphate precipitate containing 5–30 µg vector for 4–6 h

(37), washed twice in HEPES-buffered saline (10 mmol/l HEPES, 150 mmol/l NaCl,

pH 7.4), and cultured for a further 24–72 h in DMEM before harvesting. HIT cellswere transformed in the same way using an additional glycerol shock step (2 min

in 10% [vol/vol] glycerol) after the initial incubation with the DNA precipitates. Cells

were harvested using a nonenzymic procedure (Gibco cell dissociation buffer;

Gibco, Grand Island, NY), rinsed in several changes of 0.3 mol/l sucrose, 10

mmol/l MES K +, 2 mmol/l EGTA, 1 mmol/l MgSO4

, pH 6.5, and homogenized in 1

ml of the same medium using a Potter Elvejhem Teflon-on-glass homogenizer fol-

lowed by 12 passages through a 27-gauge syringe needle. The homogenate was

centrifuged at 8,000 g for 6 min and the supernatant was subjected to further cen-

trifugation at 214,000 g for 30 min in a Beckman TLN-55 rotor (Beckman, Fuller-

ton, NY) to obtain a particulate (microsomal) fraction, which was resuspendedin 300–500 µl homogenization medium (approx 0.3–1 mg/ml protein). The super-

natant was retained for-galactosidase assays. NEDH rats fed ad libitum provided

insulinoma and liver samples. Pancreatic islets were prepared by collagenase diges-

tion (38) from 12-week-old NEDH rats and 9-week-old mice of the ob / ob or balb/c

control strain. At death, the ob / ob mice ( n = 5) were obese (45.6 ± 0.4 vs. 20.3 ±0.3 g), hyperglycemic (8.0 ± 0.3 vs. 6.3 ± 0.2 mmol/l), and hyperinsulinemic (11.0

± 0.8 vs. 4.5 ± 0.6 ng/ml). All tissues were homogenized using a Potter Elvejhem

Teflon-on-glass homogenizer but otherwise treated in an identical manner to thetissue culture cell lines. Enzyme assays were usually performed within 3 h of prepa-

ration of the tissue.

In the standard G-6-Pase assay, resuspended microsomal samples (2–20 µg pro-

tein) were incubated in a conical-well 96-well polymerase chain reaction (PCR)

plate for 30 min at 30°C in 40 µl of 100 mmol/l dimethylglutarate, 20 mmol/l Na

tartrate, 10 mmol/l EDTA, pH 6.5, containing 10 mmol/l G-6-P. The reaction was

terminated by the addition of 20 µl of 10% (wt/vol) trichloroacetic acid, and the

samples were centrifuged for 5 min at 2,000 g. Twenty microliters of the supernatant

was transferred to a 96-well enzyme-linked immunosorbent assay (ELISA) plate

and mixed with 250 µl color reagent (1 part 4.2% ammonium molybdate in 5

mol/l HCl mixed with 3 parts 0.2% malachite green and filtered after 30 min), and

the optical density at 650 nm (OD650nm) was measured using Na H2PO4 (1–20

nmol) as standard. The hydrolysis of other sugar phosphates and inorganic

pyrophosphate was determined by substitution of G-6-P with 3–10 mmol/l of respective substrates. The p-nitrophenol phosphatase (p-NPP) assay was con-

ducted in a 96-well ELISA plate using 100 µl of the same buffer containing 10

mmol/l p-NPP as substrate. The reaction was terminated after 10–15 min by the

addition of 150 µl of 1 mol/l Na 2CO3, and the OD420nm was measured against o-nitro-

phenol (20–200 nmol) as standard. Glucose phosphotransferase activity was

determined in 100 mmol/l acetate buffer (80 µl), pH 5.5, containing 10 mmol/l car-

bamyl phosphate and 180 mmol/l D-glucose. Samples in a conical-well 96-well PCR

plate were incubated for 30 min at 30°C, the reaction was stopped by heating at

50°C for 3 min, and the plate was centrifuged for 5 min at 2,000 g. Samples of the

supernatant (50 µl) were transferred to an ultraviolet-transparent ELISA plate,

mixed with 200 µl of 50 mmol/l Tris buffer, pH 8, containing 3 mmol/l MgCl2, 0.2

mmol/l NADP+, and 1 U/ml G-6-P dehydrogenase, and the OD340nm was determined

after completion of the reaction (10 min) using G-6-P (10–100 nmol) as standard.

-Galactosidase activity was determined on the postmicrosomal supernatant

(5–50 µl) in a microtiter plate assay from the hydrolysis of an o-nitrophenol -D-

galactopyranoside ester (4 mg/ml) in 100 mmol/l phosphate, 10 mmol/l KCl, 1

mmol/l MgSO4, pH 7. Protein was determined on all fractions with BCA reagent

(Pierce, Rockford, IL) using bovine serum albumin as standard.

RNA was prepared by homogenization of tissue in 6 mol/l guanidinium HCl, 8%

(wt/vol) mercaptoethanol, 2% (vol/vol) laurylsarkosinate followed by CsCl step gra-

dient centrifugation (39). PolyA + mRNA was then prepared by oligo dT cellulose

chromatogra ph y, and samples were analyzed by Northern blotting after elec-

trophoresis on denaturing formaldehyde gels. Blots were hybridized for 16 h at 42°C

in 50% (vol/vol) formamide, 5 SSPE (NaCl, Na phosphate, EDTA buffer), 5

Denhardt’s reagent, and 50 µg/ml salmon testis DNA and finally washed in 0.1SSPE,

0.1% SDS at 55°C. [32P]-radiolabeled randomly primed probes were generated from

a 1,028-bp PstI fragment (nt 109–1,137) of pSVSPORT clone 2A5 and from mouse

actin. Gel loadings were adjusted on the basis of actin probe hybridization to the

samples subject to RNA dot blot analysis. Visualization and quantitation of blots was

performed by phosphor imaging (Molecular Dynamics, Palo Alto, CA).

Reverse transcription (RT)-PCR was performed as previously described (40)

using mRNA samples extracted from mouse tissues, and using cell lines as above

and from embryonic NOD mouse pancreases prepared using Triazol. Oligonu-cleotide primer sets for profiling insulin, glucagon, somatostatin, pancreatic

polypeptide, and amylase expression were as previously described (41). Primers

for -2 tubulin were forward 5-C A ACGTC A AG ACGGCCGTGTG-3; reverse 5-

G AC AG AGGC A A ACTG AGC ACC-3; for mouse islet islet G-6-Pase–related protein

(IGRP), forward 5-TTTTACCTGCTTCTCCG ACTGTT-3; reverse 5-TAG A

G A ATTTTG A A AG A ATTG ACTCC-3; and for mouse liver G-6-Pase, forward 5-

GGTTC ATCCTTGTGTCTGTG ATTGC-3; reverse 5- A ATGCCTG AC A A

G ACTCC AGCC-3. Reactions were run for 5 min at 94°C then 35 cycles of 1 min

at 94°C, 1 min at 53°C, and 2 min at 72°C with a 10-s increment per cycle, followed

by a final 20-min extension at 72°C.

RESULTS

Cloning and expression of the IGRP. A cloned cDNA frag-ment homologous to mouse liver G-6-Pase was first identified

532 DIABETES, VOL. 48, MARCH 1999

ISLET-SPECIFIC G-6-Pase–RELATED PROTEIN cDNA


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among a series of 555 randomly selected bacterial colonies froma plasmid cDNA library prepared by subtraction of mouseinsulinoma TC3 cDNA from mouse glucagonoma TC2cDNA (34). Using the 234-bp insert (nucleotide [nt] 682–904; Fig.1) as a probe, 19 polyA + clones, including 12 full-length inde-

pendent clones, were obtained from screening 2 105

colonies of a Rip Tag mouse insulinoma cDNA library. Seven-teen of the clones were judged to be derived from the samemRNA, based on the sequence at their 5 and 3 ends (150–250bp). The remaining two differed only in having nonhomologousstretches of 10 nt at their extreme 5 ends, probably repre-senting failures in the RT reaction. The longest compositesequence (1,901 bp; Fig. 1) incorporated an ORF of 1,065 bp;(nt 63–1,127) and a polyA + tract 28 nt downstream of a con-sensus polyA + addition sequence. The deduced sequence with polyA+ addition was sufficient to account for the size of theendogenous mRNA determined by Northern blotting (2,040 bp).The first AUG codon bore a consensus Kozak motif and wasshown to be a functional start site for in vitro translation. The

presence of an in-frame upstream stop codon at nt 47 indicatedthat longer alternative translational products were not possi-

ble and that the designated AUG is the true start codon. Itwas subsequently discovered that many of the clones thatshared common 5 and 3 untranslated region (UTR)sequences were actually shorter as indicated by PCR analysisusing primers incorporating the proposed start and stopcodons as shown in Fig. 1 (forward 5-C ACC ATGG ATTTCCTTC ATAGG AGT-3; reverse 5-CTCG AGCTCTATTAGTCTTCTTGT-3). Among independent 12 clones with the startcodon, two (2A5 and G3) contained a 1-kb ORF, whereas 10were ~100 bp shorter (Fig. 2). Further PCR analysis and par-tial sequencing across the exon boundaries showed that theshorter clones were all missing exon 4 (see accompanying paper). We and others (42) have previously observed RNA splicing “defects” in insulin II clones derived from the mouseinsulinoma cDNA library, and we investigated if the aboveanomalies might relate to its tumor origin. RT-PCR analysis (Fig.2) revealed that deletions within the ORF were not only pres-ent in the related Min6 mouse insulinoma cell line but also in pancreatic islets of normal mice (balb/c) and type 2 diabeticmice (ob / ob). The proportion of the longer PCR product wassimilar in each case, accounting for 35.8, 40.8, and 39.2% of thetotal PCR product, respectively, as quantified by video densit-ometry of ethidium bromide–stained gels using National Insti-tutes of Health software. Such a distribution is consistent withthe lower relative abundance of longer ORF sequencesobtained from the library screen.

The deduced ORF of 355 aa of the clones with the long ORF

(2A5 and G3) encoded a protein of 40,685 Da of a generallyhydrophobic character. Two consensus sites for NH2-linked gly-cosylation were present (aa 92 and 286), and the protein exhib-ited a COOH-terminal consensus sequence (KKXX) of an ER-resident transmembrane protein (43,44). The hydrophobicamino acids were arranged in nine major stretches, eight of which were predicted from computer analysis (TMpredict) tospan a phospholipid bilayer as alpha helixes. Seven of thosestretches contained charged amino acids: the sequence aa 28–47 contained an Asp and Arg, aa 57–77 were interrupted by Asp and Lys residues, aa 150–172 had an Arg, aa 179–193 a Glu,aa 210–230 an Arg, aa 255–273 an Arg, aa 290–307 an Arg, andresidues 318–343 a single Lys. A shorter stretch of uninter-rupted hydrophobic residues appeared at aa 116–135. The

NH2-terminus of the protein did not bear a consensus signalsequence for import into the ER, but it is conceivable that the putative transmembrane segments could function in this regard.

In vitro translation of the long ORF sequences (clones 2A5and G3) generated a 39-kDa protein, a size consistent with the

predicted molecular weight ( M r ) (40,684 Da) of the 355-aa open reading frame (Fig. 3 A). In vitro translation of the ORFof rat liver G-6-Pase in the same vector resulted in a compa-rable level of incorporation of [35S]methionine into a proteinof similar size (predicted M r 40,055 Da). One of the shorter ORF clones (2A2), which lacked exon 4, generated a proteinof 16 kDa, consistent with a COOH-terminal truncation of the

protein as a result of a change in its reading frame.The addition of dog pancreatic microsomes to an assay

translating 2A5 cRNA resulted in the partial conversion of the translated product to a higher M r form (2 kDa larger) (Fig.3 B). The change in M r was abolished by posttranslationaltreatment with endoglycosidase H after detergent solubiliza-tion or cotranslational incubation with a competitive tripep-tide inhibitor of N -linked glycosylation (15 mmol/l Asn Tyr Thr). These results suggested that at least parts of the mole-

cule were translocated into the lumen of the ER and that it didnot have a cleavable signal peptide of significant length. The presence of microsomes did not protect the molecule from proteolytic degradation with proteinase K added after thetranslation/translocation reaction, which suggested that a substantial portion of the protein was exposed on the cytoso-lic surface or that the protein had multiple transmembrane seg-ments as predicted by topologic modeling. Such modeling predicted that the largest protected fragment would havebeen around 10 kDa. Fragments smaller than 14 kDa, however,could not be resolved using the adopted analytical system.Sequence homologies. The deduced amino acid sequence(355 aa) could be aligned, except for three small gaps, withthe sequence of mouse liver G-6-Pase enzyme (357 aa). Theoverall sequence identity was 49% (69% similarity) (Fig. 4).Highest homologies were evident in aa 57–84 (81% identity,100% similarity), aa 100–132 (76% identity, 91% similarity), aa 212–236 (68% identity, 88% similarity), aa 253–285 (66% iden-tit y, 85% similarity), and aa 320–343 (64% identity, 88% simi-larity). Four of these regions corresponded to the putativetransmembrane domains, indicating that they have a functionother than simple membrane spanning. One potential site of

N -glycosylation (aa 92) occurred in an identical position in a putative second lumenal domain in both sequences; the other site in the islet sequence did not correspond to either of thetwo other sites in the mouse liver sequence, which, in anycase, are predicted to fall on the cytoplasmic or transmem-

brane segments. Both the mouse islet and liver moleculesshowed the COOH-terminal ER membrane protein retentionmotif KKXX. Homologous sequences to the mouse liver G-6-Pase have been described in other species, and it is notablethat the overall aa identity between mouse liver and fish G-6-Pase of 54% is similar to that between the mouse liver G-6-Pase and IGRP (50%), attesting to possible early evolutionarydivergence of the islet homolog. The mammalian liver sequences show 80–90% identity.

A sequence motif that is shared between the liver G-6-Pases, bacterial vanadate-sensitive haloperoxidases, andmammalian phosphatidic acid phosphatases (45,46) was alsoconserved in IGRP, as were the specific amino acids thatconstitute the active site of the haloperoxidases and that are


S.D. ARDEN AND ASSOCIATES


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FIG. 1. Nucleotide and deduced amino acid sequence of the

cloned G-6-Pase–related protein from mouse insulinoma.

The sequence was compiled from the complete bidirectional

sequencing of clone 1G3 and partial sequencing of the other

independent 11 clones indicated. Three linear motifs (45,46)shared by G-6-Pase, the type 2 phosphatidic acid phos-

phatases, bacterial acid phosphatases, haloperoxidases, and

the D r o s o p h i l a developmental protein Wunen are under-

scored with a single line, the polyA addition signal by a dot-

ted underscore, and the endoplasmic reticulum membrane

protein retention motif by a double underscore. Residues of

the catalytic site of the soluble haloperoxidases and phos-

phatases are boxed. The position of potential sites of NH2-

linked glycosylation are marked (#), and the boundaries of

the alternatively spliced form in which exon 4 is deleted (|>>

to >>|) is indicated. The deduced amino acid sequence of

exon 4 variant that results in a premature stop codon is

shown under the main sequence. The sequence information

has been deposited with the European Bioinformatics Insti-

tute (accession number Z47787 MMG6PASE submitted

19/1/1995). ¥, stop codon.


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postulated in phosphatases to interact with the substrate phosphate group. Somewhat weaker homology (27% identityover 166 aa) was seen between the islet sequence, the maizeG-6-P/Pi co-transporter (AF020813), and the related family of mitochondrial and chloroplast transporters of phosphory-lated glycolytic intermediates. This homology included a number of the residues that are highly conserved betweenIGRP and the liver G-6-Pase, including some but not all of the postulated key catalytic residues (Lys72, Pro80, Ser 167, Arg168).The homology could be indicative of an intrinsic transportactivity of IGRP/G-6-Pases.Tissue distribution of islet IGRP. Northern blot analysesof various mouse tissues revealed a single 2,040b mRNA which was highly restricted in its tissue distribution to pan-creatic islets and associated cell lines, particularly those of-cellorigin (Fig. 5 A). Phosphorimager quantitation showed that thesignals generated with the 2A5 probe with TC3 insulinoma andTC2 glucagonoma total RNA were 12 and 4% of that withan actin probe of similar specific radioactivity at the samelevel stringency of hybridization and washing, indicating a rel-atively high abundance of the mRNA. The IGRP/actin mRNA ratio in islets and Min6 insulinoma cells were higher relativeto TC3 cells (Fig. 5 B), possibly reflecting their higher state

of differentiation. In contrast, no signal was obtained frommouse testis, kidney, muscle, liver, lung, spleen, brain, pitu-itar y, gastric fundus, or heart. A number of cell lines, includ-ing those which commonly express -cell–related genes(such as AtT20 and PC12 cells), were likewise negative onNorthern blot analysis (34).PCR analyses. The remarkable -cell specificity of IGRP wasconfirmed by RT-PCR analysis of the same tissues used in theNorthern blot analysis and extended to include other tissuesand cell lines, notably other neuroendocrine tissues. A PCR product of the expected size and comparable signal obtainedwith -TC3 cells could also be obtained from theglucagonoma cell line TC2, although a 20-fold larger sampleof the starting cDNA was required to generate a signal com-

parable to that with the -cell tumor lines TC3 and Min 6.Other mouse cell lines and tissues gave negative results, par-alleling the findings with Northern blot analysis. RT-PCRreactions performed for primers for the mouse liver G-6-Pase generated a fragment of the expected size in liver andkidney but not from any of the mouse pancreatic endocrinecell lines, which either gave no product or a weak band of lower electrophoretic mobility.

Examination of expression of the cloned sequence inmouse embryonic pancreas indicated initial onset of expres-sion around day 12 and prominent expression from day 14onward, when the majority of endocrine and exocrine mark-ers were detected (Fig. 6). The pattern of expression of insulin was remarkably similar to the IGRP; glucagon expres-sion, on the other hand, was more prominent at an earlier embryologic age.Endogenous islet G-6-Pase activity and its subcellularlocalization. Studies of the catalytic properties of the insuli-noma G-6-Pase were carried out in parallel with those onthe liver microsomal enzyme prepared from a normal animalof the same strain (NEDH). Assay by either the spectropho-tometric assay (Pi release) or the radiochemical assay withthe addition of -glycerophosphate gave similar numeric

results, indicating that the hydrolysis of G-6-P was not attrib-utable to lysosomal acid phosphatase and plasma membranealkaline phosphatase activities. Subsequent studies were per-formed on a microsomal preparation that was pelleted at100,000–200,000 g after initial removal of lysosomes by cen-trifugation at 8,000 g. Judged on relative rates of mannose6-phosphate versus G-6-P hydrolysis, the liver activity exhib-ited 33% latency, the islet 55%.

Both the liver and insulinoma preparations showed broad pH dependence but differed in that insulinoma activity peaked between pH 5.5 and 6.0, and that of liver, between pH6.0 and 6.5. The K m value for the liver enzyme activity wasdetermined as 2 mmol/l, whereas the insulinoma G-6-Paseactivity could be modeled by two activities with K

m values of



FIG. 2. RT-PCR analysis of RNA splicing variants

of the IGRP in islets and islet cell lines. Samples

of oligo dT reverse transcribed total RNA were

amplified for 30 cycles using primers that

spanned the complete ORF. The exon 4 cDNA

clone 2A2 and the full-length clone G3 were

used as reference templates. Controls include

reactions performed without template, cDNA

from COS 1 cells, and reactions from which RT

was omitted (–). The results indicate that the

exon 4 transcript is the predominant form in

normal islets, diabetic islets, and insulinoma

cell lines.


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2 and 8 mmol/l (four experiments). Both liver and insulinoma activities were inhibited by 1 mmol/l chlorogenic acid (38vs. 47%; two experiments), 1 mmol/l hydroxynitrobenzalde-hyde (81.6 vs. 69.4%; two experiments), and AlF4– (48 vs. 93%;three experiments), whereas 5 mmol/l tosyl phenylalanylchloromethyl ketone was effective only with the liver enzyme(51.5 vs. 0%). The specific activity of G-6-Pase in liver micro-somal preparations was fourfold higher than in an equivalentpreparation of insulinoma microsomal membranes (Table 1).The activity measured in rat islet homogenates was lower than the insulinoma microsomal preparation but of the sameorder as insulinoma homogenates. Overnight fasting of animals

did not markedly affect the islet activity; however, culturingthe islets overnight in 5.6 or 16.7 mmol/l glucose significantl yincreased the G-6-Pase specific activity. Isopycnic density gra-dient centrifugation analysis of the postnuclear supernatant of a rat insulinoma homogenate using Percoll in isotonic sucroseas the medium showed that the major peak of G-6-Pase activ-ity paralleled the distribution of the ER marker, NADPHcytochrome c reductase, but differed from that of the lyso-some, secretory granule, and mitochondrial markers (data not shown).Enzymatic activity of the cloned sequence. Ex pressionstudies were performed with theTC3 full-length clones, 2A5and G3, and an exon 4–deleted sequence, clone 2A2. A rangeof vector systems (pCDNA, pSVSPORT, and pBK Rous sar-

coma virus [RSV]), cell types (COS 1, HIT, Min6, INS-1, AtT20,and WIF-B) and transfection protocols [Ca 3(PO4)2, lipofecta-mine, Superfect, and electroporation] were examined in thecourse of these experiments. A full-length clone of rat liver G-6-Pase served as a positive control, and the efficiency of trans-fection was evaluated by cotransfection of a pSVL constructof -galactosidase. Further controls included mock-trans-fected cells and cells transfected with an irrelevant protein.None of these protocols resulted in detectable G-6-Phydrolytic activity from the IGRP clones (Table 2). Transfec-tion of the liver enzyme, on the other hand, resulted in up to50-fold increase in G-6-P hydrolysis over basal activity.

Cotransfection of cells with plasmid encoding the liver enzyme (3.3 µg pcDNA construct) with 2A2 alone, 2A5 alone,or 2A2 plus 2A5 produced rates of G-6-P hydrolysis that were104.3 ± 10.8, 126.2 ± 11.6, and 78.5 ± 12.3%, respectively, of therates with liver enzyme alone (four experiments). Variation of assay conditions including different buffers, different pH val-ues, addition of chelating agents, or different divalent cationsdid not elicit activity from cells transfected with 2A2 or 2A5.The possibility that the islet clones were phosphatases of a dif-ferent specificity was investigated using a range of sugar phosphates (Table 3). The transfected liver enzyme showedrobust activity with a number of these substrates, indicatinga broad substrate specificit y. The rates of hydrolysis of thegeneric phosphatase substrates, p-NPP and pyrophosphate,



FIG. 3. In vitro translation of the cloned cDNA.

A: T7 polymerase RNA transcripts from the

pCDNA3 clones of the exon 4 islet clone

(2A2), the full-length islet clone 2A5, and a rat

liver G-6-Pase clone were translated in rabbitreticulocyte lysates. B: SP6 RNA polymerase

transcripts of the full-length clone pSVSPORT

2A5 were translated in rabbit reticulocyte

lysates in the presence or absence of dog pan-

creatic microsomes (MM) and subsequently

treated with proteinase K in the presence or

absence of detergents to determine the extent

of protection afforded by translocation of the

product into the lumen of the ER. Postincuba-

tion digestion of the product with endoglycosi-

dase H or addition of a competing tripeptide

(Asn Tyr Thr) acceptor of NH2 g l y c o s y l

residues during the incubation was used to

asses the extent of NH2-linked glycosylation.

A

B


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FIG. 4. Alignment and predicted membrane topology of the islet cDNA with liver G-6-Pases. A: The predicted aa sequence of IGRP clone G3

was aligned using CLUSTAL with that of the African cichlid fish, Haplochromis nubilis (AF008945), mouse liver (U00445), canine liver

(U91844), and human liver (U01120). Putative transmembrane segments are shaded and conserved charged residues within them designated

+/–. Consensus sites for NH2-linked glycosylation are also shown (#) below the sequence block. Residues defined as being of key catalytic impor-

tance in haloperoxidases and related phosphatases are boxed, as is the COOH-terminal endoplasmic reticulum retention signal. Point muta-

tions in the human liver enzyme that give rise to type 1 glycogen storage disease are indicated by the depiction of the mutant residue in ital-

ics above the sequence block. B: The predicted membrane topology is shown together with the approximate locations of the transmembrane

charges and catalytically important residues. The positioning of the catalytic residues in the juxtamembrane region of the second, third, and

fourth loops suggested close association between these regions in the tertiary structure.

A

B


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were not altered in 2A2- or 2A5-transfected COS 1 or HITcells, although they appeared to be good substrates for the liver G-6-Pase. IGRP-transfected cells likewise did not displayincreased glucose phosphotransferase activity as seen with theliver G-6-Pase. Northern blot analysis of COS 1 and CHO cellsusing either pCDNA or pBK RSV vectors demonstrated that theliver G-6-Pase and the 2A5 clone were transcribed at approx-imately equivalent levels (data not shown). The CMV pro-moter appeared more efficient than the RSV promoter in thisregard, which paralleled the level of expression of enzymaticactivity of the liver G-6-Pase. These data and the observationthat the liver G-6-Pase and IGRP are translated to a similar extent in vitro suggests that the negative results with IGRP didnot result from a failure in transcription or translation. Analy-sis of COS 1 subcellular fractions other than the microsomalfraction did not reveal the presence of G-6-Pase activity after transfection with clone 2A5, indicating that the failure to

observe activity was not the consequence of intracellular mis-targeting of the enzyme.

D I SC U SS I O N

A number of previous studies have concluded that the pan-creatic islets possess G-6-Pase activity with similar, althoughnot identical, kinetic properties to the liver enzyme(21,22,29,47,48). There are, however, some inconsistencies inthe data that may reflect technical difficulties relating to theassay procedure, tissue disruption procedures, stability of theenzyme in homogenates and intact cells, or species differ-ences. Waddell and Burchell (29) reported specific activitiesin islet microsomes at least 10-fold higher than in liver prepa-rations and attributed the activity to the liver isotype,



FIG. 5. Northern blot analyses. PolyA + mRNA samples (5 µg) ( A) or

total RNA samples (25 µg) ( B), from the indicated mouse tissues and

cell lines were hybridized to a probe prepared from a 1,028-bp frag-

ment from IGRP clone 2A5 (nt 109–1,137) and subsequently with a

mouse actin probe.

FIG. 6. RT-PCR of IGRP during embryonic development. Analyses

were performed on total RNA preparations (0.1 µg) of fetal mouse pan-

creas dissected at the indicated embryonic days. PCR was conducted

for 30 cycles using primers for representative endocrine and exocrine

pancreatic markers and the 3UTR of mouse IGRP (857 bp) sequence.

2 tubulin is used as a limiting target sequence to calibrate the mRNA

recovery and PCR amplification eff ici en c y. Samples were analyzed by

electrophoresis in 7.5% acrylamide 0.8% N , N -bisacrylamide gels run

in TBE buff e r.


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although Western blotting showed that the content of theliver G-6-Pase protein in islet and liver microsomes wasapproximately equal. The majority of other studies havereported islet enzyme activities that are 10-fold lower thanli ver. The procedures used in our experiments producedmembrane-associated activity that could be distinguishedfrom lysosomal acid phosphatase, since it hydrolyzed G-6-Pin the presence of-glycerophosphate, was unstable to mostdetergents below their critical micellar concentrations, andinactivated at pH 5. This was true for the endogenous islet andliver activities as well as the activity induced by transfectionof COS 1 cells with the cloned liver G-6-Pase. The specificactivity of G-6-P hydrolytic activity in islet homogenates or insulinoma microsomes was lower, by a factor of 3 to 10, thanin liver microsomal preparations prepared by the same pro-cedures, which could not be accounted for by a high degreeof latency or poor stability. The endogenous rat islet activityexhibited a higher K m than the liver microsomal preparationand displayed different inhibitor profiles and pH optima, sug-gesting the presence of a different catalytic activity. Westernblot analysis performed with antibodies to the liver enzyme(gift from A. Burchell) indicated at best very low expressionof the liver G-6-Pase in rat islets, which was not commensu-rate with the enzymatic activity detected in the tissue. Like-wise, we were unable to detect the liver G-6-Pase in islets or insulinoma tissues by RT-PCR (Fig. 7).

This impression, that the islet may possess a kinetically andimmunologically distinct form of G-6-Pase activity from thatof liver, provided impetus to the further cloning and charac-terization of the islet homolog of liver G-6-Pase, which wedetected previously during the characterization of a-cell–enriched cDNA library (34). The cDNA fragment of theORF that was used as a probe for this study was originallyidentified among 550 clones that were randomly selectedfrom cDNA restriction fragments of a TC3 cell line cDNA subtracted with an equivalent preparation from TC2 cells.Comparison of the cloned full-length islet sequence with thedatabase further revealed that five other clones, classified as“unknown” in the original library screen, were also derivedfrom the 3 UTR of the IGRP-related sequence (accession

numbers Z47768 and Z47769). The frequency of these clonesin the subtracted library was of the same order as thatobserved for islet-associated polypeptide (amylin), the pro-hormone processing enzyme PC1, calbindin, and neuropep-tide Y, which placed it among the -cell–enriched proteinsexpressed at moderate to high levels. The Northern blot (Fig.5) and RT-PCR (Fig. 7) analyses performed in the presentstudy confirm the results of the previous mRNA dot blotanalyses with clone Z47768, which demonstrated a moderatelevel of expression and remarkable restriction of the mRNA sequence to the -cell (34).

In vitro translation experiments indicated that the bulk of theIGRP sequence is inserted co- or posttranslationally into ERmembranes and that it probably transverses the membrane sev-eral times. The presence of the COOH-terminal KKXX reten-tion/retrieval motif is consistent with an ER-localized proteinand the finding that insulinoma G-6-Pase activity, like the liver enzyme, is expressed in the ER. By analogy to other ER-resi-dent membrane proteins, the COOH-terminus would need toface the cytosol to be functional. The conserved N -gl ycos yla-tion at aa 92 would be expected to face the ER lumen, as

would the putative catalytically important residues (Fig. 4). Theeight-transmembrane spanning model predicted by the pro-gram TMPredict, which is based principally on secondarystructural considerations, is inconsistent with these infer-ences, as is a previous six-transmembrane model (6). However,the islet sequence can be accommodated in a nine-transmem-brane model, although the putative 1st and 7th membrane-spanning alpha helices are less hydrophobic in the isletsequence, while other essential features are conserved. Whatis unusual in the alignment of the cloned sequence with the liver isotypes is the high degree of sequence conservation of the longhydrophobic stretches. This argues for a conserved biochem-ical function as well as a structural organization. The additional

presence of conserved charged residues in these segments isconsistent with a function either as partially buried catalytic siteor as a membrane ion channel, as previously proposed for theliver enzyme.

The conservation of primary sequence, membrane topol-og y, and gene structure (see following paper) between thecloned islet sequence and the liver G-6-Pases argue in favor of its being an active islet-specific isoform of G-6-Pase. Theexistence of such an activity would explain some of the dis-crepancies between the kinetic and immunologic reactivityof islet versus liver enzyme and would have important impli-cations for the regulation of glycolytic flux and stimulus-secretion coupling of glucose-induced insulin secretion. Wewere, however, unable to demonstrate such activity in the cur-

rent experimental series. A number of possible explanationsfor this failure are discussed below in the context of subse-quent experiments aimed at revealing the biochemical prop-erties of the protein.

The liver enzyme is part of a multimolecular functional unitincorporating the catalytic subunit, transporters for G-6-P(T1) (18), phosphate (T2) (49), and glucose (T3) (50) and a 20-kDa stabilizing protein (51). All these proteins have beencharacterized at the molecular level. There is little informa-tion on the physical interaction of the subunits and their rel-ative stoichiometry. If the IGRP requires integration into a complex to be active, it is conceivable that the other subunitsmight not be provided in the COS or HIT cell lines used hereor that IGRP is associated with specific isoforms of these pro-



TABLE 1Endogenous G-6-Pase activity in pancreatic islet tissue insulinoma and liver

G -6 - Pa s eSample (nmol · min–1 · mg–1)

Mouse islets 2.6 ± 0.3 (5)Rat islets

Fed animals 6.5 ± 1.4 (6)24-h starved animals 7.0 ± 1.0 (6)Cultured in 5.6 mmol/l glucose 18.0 ± 2.6 (4)Cultured in 16.7 mmol/l glucose 16.8 ± 5.3 (4)

Insulinoma microsomes 33.1 ± 6.4 (12)Liver microsomes 132.7 ± 24.9 (9)

Data are means ± SE (number of experiments). Batches of 200–400 islets were prepared by collagenase digestion and either used fresh or cultured overnight with the indicated glucose con-centrations in DMEM. Tissues were homogenized and subjectedto differential centrifugation to obtain a microsomal fraction( 2 5 , 000 – 1 0 0 ,0 0 0 g p e l le t ) .


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teins. The catalytic subunit of liver G-6-Pase, however, hasactivity in disrupted microsomes when coupled to mutant,functionally inactive transporters or in the presence of their inhibitors, chlorogenic acid or hydroxy nitro benzaldehyde.In our experiments, the full-length liver catalytic subunit dis-

played robust hydrolytic function in the SV40-transformedhamster insulinoma cell line HIT, which suggested either norequirement for these subunits or the fact that the insuli-noma accessory proteins, if different, were compatible withthe liver G-6-Pase. Neither the transfected liver activity nor the islet activity showed marked latency in our assays asindicated by similar rates of mannose 6-phosphate andG-6-P hydrolysis, which in itself would tend to precludedependence on coexpression of transporter molecules for activity in our assays.

The observation that both the liver and islet cDNAsencode transmembrane proteins with similar topologic fea-tures to membrane transporters and channels suggested thatthe catalytic subunit might function only within homotypic or heterotypic complexes capable of transport of the substrate

to the active site. Cotransfection of the IGRP with the liver G-6-Pase resulted in rates of G-6-P hydrolysis that were no dif-ferent from that with the liver catalytic subunit alone. Like-wise, the possibility that an active islet complex is gener-ated by the association of the full-length molecule with thetruncated exon 4 deletion mutant appeared precluded by thefailure of cotransfection to generate activity.

The conservation of amino acids implicated in phosphatase activity between the liver G-6-Pase and IGRP arguesin favor of the islet sequence having phosphohydrolase activ-



TABLE 3

Phosphohydrolase and phosphotransferase activities of liver and IGRP transiently expressed in COS 1 cells using the pCDNA3 vector

Empty vector Liver G-6-Pase Clone 2A5A s s a y (nmol · min –1 · mg–1) (nmol · min–1 · mg–1) (nmol · min–1 · mg–1)

10 mmol/l G-6-P 2 4 . 3 0 7 72. 0 9 3 4.4 010 mmol/l mannose 6-phosphate 8 .36 7 5 8 . 36 17 . 5 810 mmol/l pyrophosphate 8. 6 1 2 9 2.6 1 9 . 0 310 mmol/l 3-phosphoglycerate 0. 8 7 2 . 3 9 0 .343.3 mmol/l glyceraldehyde 3-phosphate 6 4 .91 1 2 6 . 97 7 6 . 3 910 mmol/l phosphoenolpyruvate 10 . 5 7 6 5 .2 5 1 3 . 772.5 mmol/l xylulose 5-phosphate 1 0 . 95 10 . 3 4 9 . 2210 mmol/l ribose 5-phosphate 5 . 3 6 145. 0 3 1 2.2 55 mmol/l ribulose 5-phosphate 9 .71 1 2 8 . 19 22 . 4 910 mmol/l 6-phospho gluconate 0. 8 7 2 0 . 63 0.5 64 mmol/l glucose-1-phosphate 0 .50 0. 5 1 0 . 6 35 mmol/l glucose-1,6-bisphosphate 0 . 5 5 2 . 08 0. 4 15 mmol/l fructose-1-phosphate 3 .3 0 6 1 . 6 6 2 .4 310 mmol/l fructose-6-phosphate 1 4. 8 9 4 4 2 .2 8 1 3 . 8810 mmol/l fructo-1,6-bisphosphate 2 . 1 3 1 3. 8 0 2 . 3 95 mmol/l mannose 1-phosphate 0. 3 9 0 . 96 0. 3 310 mmol/l p-NPP 461 . 1 7 874 . 8 6 4 74. 1 510 mmol/l p-NPP + 10 mmol/l G-6-P 4 9 4 . 43 66 6 . 5 0 456 . 0 7Glucose phosphotransferase 2 .2 0 1 0 5 0. 2 8 7 . 5 0

Data are means of duplicate analyses from two separate transfection experiments. Phosphohydrolase activities were determined under standard G-6-Pase assay conditions at the indicated substrate concentrations, and release of Pi was determined spectrophometrically.p-NPP was determined spectrophometrically from the release of p-nitrophenol and assayed in the absence or presence of G-6-P as a competing substrate. Phosphotransferase activity was determined in the presence of 10 mmol/l carbamyl phosphate and 180 mmol/lglucose, and the product (G-6-P) was determined enzymatically.

TABLE 2Transient transfection analysis

HIT cells ( n = 5) COS 1 cells ( n = 7)

G - 6- P ase -Gal a c t o s id a s e G - 6 -P a s e - G a la c to s i daseTr a nsf e ct a nt (nmol · min–1 · mg– 1) (nmol · min–1 · mg– 1) (nmol · min–1 · mg– 1) (nmol · min–1 · mg– 1)

Empty vector 5.29 ± 1.60 38.9 ± 17.5 3.48 ± 0.78 572 ± 334

Islet 2A2 3.65 ± 0.50 26.7 ± 10.8 5.51 ± 1.86 1,215 ± 389Islet 2A5 3.18 ± 0.21 47.4 ± 21.7 5.97 ± 1.67 846 ± 387Liver G-6-Pase 28.70 ± 7.25* 23.4 ± 8.3 101.4 ± 22.3† 894 ± 400

Data are means ± SE. Cells were transfected with a combination of 30 µg pCDNA3 vector containing the indicated construct alongwith 5 µg pSV -galactosidase construct as a transfection control. Cells were harvested after 48 h, and the enzyme activities weredetermined in either a postmitochondrial microsomal pellet (G-6-Pase) or postmicrosomal supernatant (-galactosidase). LiverG-6-Pase activity was significant at * P < 0.02 in HIT cells and † P < 0.005 in COS cells.


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it y, though not necessarily directed at G-6-P. Similar motifs,for example, exist in bacterial -glycerophosphatases andapyrase that show little overall sequence homology to the liver G-6-Pase or IGRP. None of the sugar phosphates and gly-colytic intermediates that were shown to be substrates of liver G-6-Pase in transfection experiments appeared to behydrolyzed by IGRP. These experiments confirmed that theliver G-6-Pase has a remarkably broad substrate specificit ywhen the ER membrane is disrupted, and access to the catalyticsite does not depend on specific transporters. LiverG-6-Pase, but not IGRP, was shown to hydrolyze the generic phosphatase substrate p-NPP, a finding that would indicatethat the islet protein possesses no phosphatase activity. Like-wise, neither the well-documented pyrophosphatase and glu-cose phosphotransferase activity of the liver enzyme could be

observed in cells transfected with IGRP. The phosphatase motif that is conserved in the islet and liver G-6-Pase isotypes is alsofound in the bacterial chloroperoxidase; however, cells trans-fected with either the liver G-6-Pase or IGRP showed no activ-ity in a standard haloperoxidase assay determining the bromi-nation of phenol red in the presence of H2O2 (data not shown).

Ten of the 12 islet cDNAs defined as full-length on thebasis of their 5 sequence were found to be missing exon 4 intheir cDNA. This led to the hypothesis that there may bestrong selective pressure against IGRP retaining enzymaticactivity in the tumor from which it was cloned. This couldoccur as a consequence of the documented upregulation of low K m hexokinase (26,52) which, in the presence of anactive G-6-Pase, could lead to excessive intracellular ATP

hydrolysis incompatible with cell survival and growth. How-e ver, RT-PCR analyses from normal and genetically obesemouse islets showed that predominance of the exon4–deleted form occurs normally. The possibility that IGRPmight have gained inactivating point mutations duringcloning received some credence from the initial observationthat IGRP differed in sequence from liver G-6-Pase at positionsSer

120

and Lys209

, where point mutations (Ala →Thr andLeu→Pro, respectively) have been found in two cases of type 1 glycogen storage disease (13). It is not clear, however,that such changes would lead to an inactive enzyme in the present case. Moreover, the genomic sequence reported in theaccompanying paper confirms the cDNA sequence of theORF determined here. This also precludes the possibilitythat inactivating somatic mutations might have occurred asa result of tumorigenesis leading to the generation of theinsulinoma cell line.

It is conceivable that IGRP is a pseudogene. Against this idea is the finding that the gene is transcriptionally and transla-tionally active and that its expression is highly tissue-specific,which suggests strong conservation of cis-acting regulatory

elements. Although the islet and liver sequences appear tohave diverged early in vertebrate evolution, it is noted that theregions of the coding sequences that are strongly conservedbetween the fish and human liver homologs are those mosthighly conserved between the liver G-6-Pase and IGRP. Thisincludes individual amino acids implicated in the catalyticmechanism of the phosphatases as well as functional domainsinvolved in translocation of segments of the polypeptide chaininto the lumen of the ER. The final verdict on whether the isletsequence is truly inactive will require investigation of itsexpression at the protein level. The latter approach has so far been impeded by the difficulty in raising specific high-titer antibodies to peptide sequences or recombinant forms of the protein. It remains a possibility that IGRP is either poorlytranslated in a cellular context or is degraded more rapidly thanthe equivalent liver G-6-Pase. Endogenous islet G-6-Pase activ-ity is reported to disappear within an hour of tissue culture (29),a finding that may have some bearing on the present results andthe variability in previous assays of islet activity. Notwith-standing the failure to demonstrate a biochemical functionfor the cloned sequence, the availability of the IGRP cDNA will

permit further investigation of its transcriptional regulation inconditions where glucose responsiveness of the pancreaticislet is altered in a physiologic or pathologic context. Theappearance of the enzyme in parallel with insulin expressionduring development further emphasizes the fact that the tissue-s pecific expression of the protein and its regulation may be

closely tied to insulin gene expression and share commontranscriptional activation mechanisms. The cloning and char-acterization of the encoding gene and identification of the pro-moter as described in the following article presents further opportunities for study of the enzyme and its function bygenetic manipulation in animals.

AC KN O WL E D GM E N TS

This work was supported by the Children’s Diabetes Foundation.Dr. Gerhard Christofori (Biozentrum, Vienna, Austria) is

thanked for the provision of the pSVSPORT Rip Tag insulinoma cDNA library. Dr. George Giddes (Hormone Research Institute,University of California, San Francisco) for mouse embryonic

pancreas samples, Dr. Anne Burchell (Obstetrics & Gynecology,



FIG. 7. RT-PCR analysis of tissue-specific expression of IGRP and

liver G-6-Pase. Analyses were performed on the total RNA preparations

(0.1 µg) of the indicated tissue for 30 cycles using primers designed

to amplify the 3UTR of either the mouse liver G-6-Pase (522 bp) or

mouse IGRP (857 bp) sequence. Samples were analyzed by elec-

trophoresis in 1.5% agarose gels run in TBE buffer.


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Dunned) for anti-liver G-6-Pase antibody and helpful discus-sion in the initial phase of this project, and Carrie John for assis-tance in preparation of the manuscript.

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