functional expression of the rat pancreatic islet glucose

0013.7227/95/$03.00/0 Endocrinology Copyright 0 1995 by The Endocrine Society

Vol. 136, No. 10 Printed in U.S.A.

Functional Expression of the Rat Pancreatic Islet Glucose-Dependent Insulinotropic Polypeptide Receptor: Ligand Binding and Intracellular Signaling Properties*

MICHAEL B. WHEELER?, RICHARD W. GELLING?, CHRISTOPHER H. S. MCINTOSH, JOHN GEORGIOU, JOHN C. BROWN, AND R. A. PEDERSON

Departments of Medicine and Physiology, University of Toronto, Toronto, Ontario; and the Department of Physiology, University of British Columbia (R. W.G., C.H.S.M., J.C.B., R.A.P.), Vancouver, British C!olur”nbia, -&nada -

ABSTRACT Incretins are endogenous peptides released from the gastrointes-

tinal tract into the circulation during a meal that potentiate glucose- stimulated insulin secretion. At present, there are two established incretins: glucose-dependent insulinotropic polypeptide (GIP) and the truncated glucagon-like peptides (tGLPs), which are now being investigated for use in the treatment of diabetes mellitus. In the present study we cloned a rat islet GIP receptor complementary DNA (GIP- Rl) to answer several important questions regarding the ligand-binding and intracellular signaling properties ofthe GIP receptor. GIP-Rl, when expressed transiently in monkey kidney (COS-7) or stably in Chinese hamster ovary (CHO-Kl) cells, demonstrated comparable high affinity binding for either synthetic porcine (sp) GIP or synthetic human (sh) GIP. The IC,, values for displacement of [1251]spGIP in CHO-Kl cells were 2.6 + 0.8 and 3.1 2 0.9 nM for two different preparations of shGIP, and 3.7 2 1.5 and 3.6 2 0.4 no for two preparations of spGIP. Saturation isotherms obtained with both intact cells and membranes gave monophasic binding curves with apparent & values of 204 i 17 and 334 2 94 PM, respectively. Cells expressed 12-15 X lo3 receptors/cell. In COS-7 cells, spGIP and shGIP also exhibited similar IC,, values (7.6 + 1.2 and 8.9 -C 1.8 nM, respectively). The receptor in CHO-Kl cells bound GIP-(l-30) with lower affinity (IC,, = 39 ? 17 nM), whereas the fragments GIP-(19-301, GIP-( 18-28), and GIP-(21-26) showed no apparent binding. The specificity of the receptor was further examined using several structurally related peptides. Surprisingly, exendin-(9-39) [Ex-(g-39)1, a GLP-1 receptor antagonist, and Ex-4-(l-39), a GLP-1 receptor agonist, demonstrated some affinity for the GIP receptor, with 39% and 21% displacement of [ 1251]spGIP, respectively, at 1 pM. Other members of

the secretin/vasoactive intestinal peptide family of peptides tested showed no interaction. GIP-Rl receptor binding correlated with activation of the adenylyl cyclase system, whereby spGIP and shGIP evoked concentration-dependent increases in CAMP accumulation with EC,, values of 8.7 ? 1.5 X lo-I’M and 8.1 ? 1.6 X lo-I'M for spGIP and shGIP, respectively. Increases in CAMP in the presence of 10 nM spGIP were not dependent on the ambient glucose concentration, with 22- and l&fold increases in CAMP accumulation at 0.1 and 5.5 mM glucose, respectively. Despite its ability to displace [12511sp- GIP, Ex-(9-39) was unable to act as a GIP receptor antagonist at the concentration tested (1 PM), a concentration that elicited a 65% reduction in tGLP-l-stimulated CAMP accumulation in cells expressing the tGLP-1 receptor. A biphasic increase in intracellular Ca2+ ([Ca2+],) was also elicited by spGIP (50 nM), with an acute transient phase (Pl; A[Ca2+li = 114 t 8 nM) followed by a sustained elevation (P2; A[Ca”l, = 36 -’ 6 nM). Pl was attributed to the mobilization of an intracellular thapsigargin-sensitive [Ca2+li pool, whereas P2 rep- resented Ca2+ influx through a cation channel that was not affected by the L-type Ca2+ channel blocker, nifedipine (10 PM). This study establishes for the first time that pancreatic islets express a receptor with high affinity for the powerful insulinotropic polypeptide, GIP. This receptor, when expressed in either COS-7 or CHO-Kl cells, does not display preferential affinity for either human or porcine GIP preparations and can activate two potential intracellular triggers for insulin secretion, CAMP and [Cazfli. The increase in [Ca2+li involves the mobilization of intracellular Ca2+ stores and the activation of a cell surface cation channel to allow Ca2+ influx. (Endocrinology 136: 4629-4639, 1995)

G ASTRIC INHIBITORY polypeptide/glucose-dependent insulinotropic polypeptide (GIP) is a 42-amino

acid peptide hormone produced by the K cells of the mammalian duodenal and jejunal mucosa and released in response to the ingestion of glucose, fat, and some amino acids

Received March 8, 1995. Address all correspondence and requests for reprints to: Dr.

Raymond A. Pederson, Department of Physiology, Faculty of Medicine, University of British Columbia, 2146 Health Sciences Mall, Vancouver, British Columbia, Canada V6T 123.

*This work was supported by grants from the Medical Research Council (MT-12898), the Banting and Best Diabetes Center, and the University of Toronto Faculty of Medicine Dean’s fund (to M.B.W.) and by the British Columbia Health Research Foundation.

t M.B.W. and R.W.G. contributed equally to this work and should both be considered as first authors.

(reviewed in Refs. 1 and 2). GIP was initially identified and isolated on the basis of its ability to inhibit gastric acid secretion (enterogastrone action) and later was shown to be a potent stimulant of insulin secretion (incretin) in the presence of hyperglycemia (reviewed in Refs. 1 and 2). GIP is now widely acknowledged as being one of two established incretins involved in the enteroinsular axis in man (1, 3,4) and other species (1,5), the other being truncated glucagon- like peptide-l [tGLP-1; GLP-1(7-36) amide and GLP-1(7-37)] (reviewed in Ref. 6). The latter incretin is now being investigated as a potential therapeutic agent in the treatment of noninsulin-dependent diabetes mellitus (NIDDM) (6 - 8).

Both high and low affinity GIP-binding sites have been demonstrated in islet-derived p-cell lines, a gastric tumor cell line (HGT-1) (reviewed in Refs. 1 and 2), and rat olfactory cortex (9), the high affinity component of which exhibited K,

4629

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4630 GIP RECEPTOR, BINDING AND SIGNALING Endo. 1995 Vol 136 . No 10

values ranging from 0.1-7 nM (9,lO). GIP has been shown to increase intracellular levels of CAMP (10-12) and Ca’+ ([Ca2’li) in tumor cell lines (11, 12) and isolated islets (13). In HIT-T15 cells, the increase in [Ca2’], was attributed to the activation of voltage-dependent Ca2+ channels (VDCC), because the L-type calcium channel antagonist, nifedipine, inhibited GIP-stimulated increases in [Ca2+], and insulin secretion (12).

Usdin and colleagues recently (14) isolated a complementary DNA (cDNA) encoding a putative seven-transmem- brane receptor protein with a high degree of similarity to other receptors in the vasoactive intestinal peptide (VIP)/ secretin receptor family. Expression studies revealed that GIP was the only candidate peptide tested that elicited a high affinity CAMP response and increased [Ca”]i in reporter cell lines (14). Interestingly, the presence of receptor transcripts was demonstrated in a number of extrapancreatic tissues by a polymerase chain reaction (PCR)-based approach. This included the brain, which had not previously been considered as a GIP target tissue. The present study was undertaken to determine whether the GIP receptor expressed in the central nervous system and RIN cells (14), which are phenotypically multipotential (15), was also expressed in normal pancreatic islets. A second objective was to characterize in greater detail the ligand-binding properties and intracellular second mes- senger systems used by the rat islet GIP receptor to transduce the GIP signal. Thirdly, it was considered to be of value to critically assess the efficacies of several available GIP preparations for receptor binding and activation. These studies are important given the controversy surrounding the insulinotropic activities of different GIP species and preparations (7,16) and the potential therapeutic value of the incretins for the treatment of NIDDM. To answer these questions we used reverse transcription (RT)-PCR to isolate a GIP receptor cDNA from rat islet RNA. It was found to be identical, except for one putative amino acid change (Gluzl-+Glnzl), to that previously isolated by Usdin et aZ. (14), and we established that this GIP receptor is expressed in pancreatic islet cells. The receptor was functionally expressed in the monkey kidney (COS-7) and Chinese hamster ovary (CHO-Kl) cell lines and used to study the receptor-binding and adenylyl cyclase- stimulating activities of different synthetic preparations of porcine and human GIP. In addition, the mode of action of GIP in increasing [Ca2+li was investigated.

Materials and Methods

Peptides

Synthetic peptides were obtained from the following commercial sources: synthetic human (sh) GIP (lot 94081641, synthetic porcine (sp) GIP (lot 033785), GLP-l-(7-37) (lot 019860), shGLP-2 (lot 008674), and VIP (lot 015174) from Peninsula Laboratories (Belmont, CA); porcine glucagon (lot G4211963) from Novo Biolabs (Bagsvaerd, Denmark); and shGIP (lot ZK887, spGIP (lot 7580, exendin-4 (Ex-4; lot ZL765), and Ex-(9-39) (lot ZL777) from Bachem California (Torrance, CA). Natural porcine (npf GIP was purified as described previously (17). shGIP- (l-30)-OH and spGIP-(17-30) were the kind gifts of Dr. N. Yanaihara (Shizuoka, Japan) and Dr. S. St. Pierre (INRS Sante, Montreal, Canada), respectively. spGIP-(19-30) was prepared by tryptic digestion of spGIP- (17-30) and purification of the major product by reverse phase HPLC (RI’-HPLC). The identity of the peptide was determined by sequence

analysis. The peptides shGIP-(18-28), shGIP-Q-26), tGLI’-l-(21-26), and glucagon-(21-26) were synthesized by solid phase techniques by the Nucleic Acid-Protein Service Unit, University of British Columbia, and purified by RP-HPLC.

Isolation and characterization of a cDNA encoding the rat GIP receptor

First strand cDNA was prepared from oligo(deoxythymidine)- primed total RNA isolated from purified rat pancreatic islets (-2000 islets, yielding -20 pg total RNA) via RT-PCR (Perkin-Elmer/Cetus, Norwalk, CT). Oligonucleotide primers corresponding to nucleotides 163-184 (5’-AGGATGCCCCTGCGGCTGTTGC-3’) and 1537-1515 (5’- GTCCTAGCAGTAACTTTCCAAGA-3’) were designed to amplify the coding region of the rat islet GIP receptor cDNA based on the published sequence of Usdin et al. (14). The amplification was carried out using an annealing/extension temperature of 62 C for 35 cycles (Perkin-Elmer/ Cetus). A single product’of the appropriate size i-1.4 kilobases) was obtained and introduced into DCRII (Invitroeen. San Dietro. CA). Clones ” ”

were subsequently introduce2 into the HindIII-XbaI sites of the expression vector pcDNA 3 (Invitrogen), and the plasmid was designated pGIP-RI. A second independent receptor cDNA was generated by RT- PCR using the same methodology and was designated pGIP-IQ. To determine whether the PCR products generated corresponded to that isolated by Usdin et al. (14), GIP-RI and GIP-R2 were mapped by re- striction analysis and partially sequenced using a T7Sequencing Kit (Pharmacia Biotech, Uppsala, Sweden). The GIP-Rl and GIP-R2 sequences differed from that previously reported (14) by only 1 nucleotide in 600 sequenced, resulting in a single amino acid difference (Glu2’+Gln2’), indicating that the two cDNAs encode homologous receptor proteins.

Cell transfection

For transient expression, COS-7 cells (2.5 X 106) were seeded in IO-cm dishes (Becton Dickinson, Lincoln Park, NT) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) ‘su&emented with 10% fetal bovine serum (GIBCO, Grand Island, NY). Cells were transfected the following day with 5 pg pGIP-RI, pGIP-R2, pcDNA3, or pGLP-Rl (pcDNA-1 containing the cDNA for the rat GLP-1 receptor) by the diethylaminoethyl-dextran method, as previously described (18). Fif- teen hours after transfection, the cells were passaged into either 24-well plates for CAMP studies or IO-cm dishes for binding studies and cultured for an additional 48 h. The cells were then detached from dishes using PBS containing 0.1 rnM EDTA and treated as described for each specific analysis. For production of a permanent CHO-Kl cell line expressing the GIP receptor, cells were grown in DMEM-Ham’s F-12 medium supple- mented with 10% fetal calf serum (GIBCO) on lo-cm dishes until con- fluent. Cells were transfected with 10 lg pGIP-Rl using the calcium phosphate coprecipitation method (5-Prime 3-Prime, Boulder, CO). In- dividual clones expressing pGIP-Rl were isolated by G418 selection (800 &ml; GIBCO) and further selected for high level expression by screening for GIP-stimulated CAMP production and [‘Z51JspGIP binding, as described below. One clone, designated rGIP-15, was selected for further characterization.

Iodination of spGIP

spGIP (5 pg; Peninsula) was iodinated by the chloramine-T method and further purified by RP-HPLC to a specific activity of approximately 250-350 FCi/pg, as previously described (19). Aliquots were lyophilized and stored at -20 C until use.

Binding analysis

Binding analysis on cells was performed as previously described (18) with minor modifications. Briefly, COS-7 cells were washed twice in binding buffer (standard DMEM containing 4 g/liter glucose unless otherwise specified, 0.5% BSA, and 0.1 me/ml bacitracin. DH 7.4) and preincubateh for 30 min at 376. Cells (5 X ‘i+/tube) were’&ubated for 30 rnin at 37 C with radiolabeled peptide (50,000 cpm; -90 PM) in the presence or absence of unlabeled peptide in a final volume of 200 ~1.



GIP RECEPTOR, BINDING AND SIGNALING

After incubation, cell suspensions were centrifuged at 12,000 X g and washed once in ice-cold binding assay buffer, and the cell-associated radioactivity in the pellet was measured in a y-counter.

Whole cell studies with CHO-Kl cells (rGIP-15) were carried out either as described above for COS-7 cells or while attached in 24-well culture plates, with minor modifications. Incubation times were 1 h at 37 C and room temperature (RT) or 4 h at 4 C. Cells were washed once with ice-cold buffer, solubilized with 0.1 M NaOH (0.5 ml), and trans- ferred to test tubes for counting of cell-associated radioactivity (18). Membranes from CHO-Kl cells stably expressing GIP-Rl were isolated and prepared for saturation and competition assays as described by Samama et al. (20). All studies using membranes were carried out at 4 C for 4 h using approximately 100 pg protein/tube. The membrane fraction protein concentration was determined using the bicinchoninic acid method (Pierce Chemical Co., Rockford, IL). Nonspecific binding for both cell types and membranes was defined as that measured in the presence of an excess of synthetic porcine GIP (1 FM), and specific binding was expressed as a percentage of the maximum binding.

Measurement of CAMP

COS-7 cells (24 h after transfection) and CHO-Kl clones expressing GIP-RI were passaged into multiwell plates and cultured for an additional 48 h. Cells were then washed in assay buffer containing 0.5% BSA and 0.1% bacitracin and preincubated for 30 min, followed bv a 30-min stimulation period with-test agents [including 1 PM isobutylmethyl- xanthine (IBMX) in COS cells] as previously described (18). Cells were extracted with 70% ethanol, and CAMP levels were measured by RIA (Biomedical Technologies, Stoughton, MA).

Rat pancreas perfusions

Perfusion studies were carried out as previously described (21). Briefly, overnight-fasted rats were anesthetized (60 mg/kg pentobar- bital), and the pancreas and associated duodenum were isolated. The perfusate was a modified Krebs-Ringer buffer containing 3% dextran iSigma) and 0.2% BSA, gassed with 95% O,-5% CO, to achieve a pH of 7.4. The GIP ureuarations were delivered as a linear gradient of O-l nM over a 50-min period in the presence of 16.7 mM glucose. The experimental procedure and insulin RIA details were previously described (21). Results are expressed as the mean integrated insulin response (nanomoles) over 50 min * SEM.

Cytosolic Ca2+ measurements

Fluorimetuu. Free cvtosolic calcium concentrations lCa’+J+ were determined using a Hitachi F2000 spectrophotometer (Hitachi, Tokyo, Japan) as ureviouslv described (22). Brieflv. COS-7 cells were loaded in DMEM with 2 WM f&a-2/AM (Molecular Probes, Eugene, OR) for 20 min at 37 C. Aliquots of these cell suspensions were washed by sedimentation, and approximately 2.5 X lo5 cells were resuspended in KRPD buffer (140 mM NaCl, 4 mM KCl, 10 mM glucose, 10 mM HEPES, and 1 mM MgCI, with or without 1 mM CaCl,, pH 7.4) and placed into a cuvette (no. 67.775, Sarstedt, Germany) with magnetic stirring in a thermostatically controlled (37 C) chamber. Fluorescence was measured at 37 C, with excitation at 335 nm and emission at 510 nm. [Ca”], calibration was performed using ionomycin and Mn*+, with a Ka of 224 nM and a fluorescence maximum/fluorescence minimum ratio of 3, as previously determined (23).

Confocul microscopy. A confocal laser scanning microscope (CLSM, Bio- Rad-600, Bio-Rad Laboratories, Richmond, CA) was used to analvze GIP-evoked Ca2+ fluxes in individual COS-7 cells transfected with pGIP- Rl. Brieflv. COS-7 cells were loaded with 10 NM fluo3/AM (Molecular Probes) for 1 h in PBS-DMEM (vol/vol), 0.5% dimethylsulfoxide, and 0.01% pluronic acid. Subsequently, cells were washed twice in DMEM, and changes in [Cazfli were analyzed in the presence of 50 nM GIP. Flu03 was excited using the 488 nm line of the argon laser, and emitted fluorescence was detected through a low pass filter with a cut-off at 515 nm. Images were collected digitally, and a false color scale was generated for quantitative purposes, where blue corresponds to lower and red to higher Ca2+ levels. The changes in fluorescence were measured using

CONRAD, a program for PC analysis and the preparation of confocal images, written by T. A. Goldthorpe, Department of Physiology, University of Toronto.

Data analysis

Binding and CAMP data were analyzed using the computerized non- linear regression analysis program PRISM (GraphPad, San Diego, CA). In the case of saturation and competition binding studies, data were analyzed for both one- and two-affinity state models. The two-affinity state model was adopted only when it described the data more accu- rately (P < 0.05) than a model for a single form. IC,, (binding studies) and EC,, (CAMP experiments) values from experiments were expressed as the mean i SEM from at least three individual experiments, except where stated in the text. Analysis of variance was performed on all IC,, values and CAMP measurements, as indicated in the text. Student’s t test was used for determination of significant differences between individual treatments or peptide preparations. P < 0.05 or less was considered significant.

Results

Characterization of GIP receptor binding

To demonstrate that the PCR-amplified cDNA isolated from rat islet tissue encoded a GIP-specific receptor, binding analyses, using spGIP and shGIP for displacement, were initially performed on COS-7 cells transiently expressing GIP-Rl. Bot!*peptide preparations inhibited the specific binding of [ I]spGIP to COS-7 cells in a concentration- dependent manner and with similar potencies (Fig. 1A). The I&a values for displacement were 7.6 t 1.2 and 8.9 + 1.8 nM for spGIP and shGIP, respectively (n 2 3). Similar displacement results were obtained with COS cells transfected with the second GIP receptor cDNA, GIP-R2, whereas no specific binding to control nontransfected COS-7 cells or to cells transfected with the expression vector pcDNA 3 or the rat GLP-1 receptor (18) (data not shown) was observed.

As stable CHO-Kl cell lines expressing GIP-Rl provided a more practical approach for the analysis of ligand binding, clone rGIP-15 was used to further characterize the GIP receptor. Saturation isotherms obtained with both intact cells and membranes gave monophasic binding curves with the apparent Kd values of 204 2 17 PM (Fig. 2) and 334 5 94 PM

(data not shown), respectively. rGIP-15 was determined to express approximately 12-15 X lo3 receptors/cell. Compe- tition assays with CHO-Kl cells were carried out to inves- tigate any possible differences in affinity for various GIP preparations and analogs. Data consistently fitted a one-site model at all temperatures examined (4 C, RT, and 37 C), although IC,, values were consistently lower at 4 C (1.2 -C 0.1 PM) than at RT or 37 C for a synthetic porcine GIP preparation. As experiments performed at room temperature gave more consistent ICsO values than those at 37 C (1.2-8.9 nM) with a convenient incubation time, all other experiments were performed at RT. Competitive binding experiments revealed no significant differences either between the mean IC,, values for porcine and human GIP [shGIP-I (Bachem), 2.6 t 0.8 nM; spGIP-1 (Bachem), 3.7 i 1.5 nM; shGIP-2 (Peninsula), 3.1 C 0.9 nM; spGIP-2 (Peninsula), 3.6 k 0.4 nM] or between GIP preparations produced by the two different commercial suppliers (P > 0.05; n = 3; Fig. 1B). npGIP had a similar IC,, value (-3 nM; data not shown), further



4632 GIP RECEPTOR, BINDING AND SIGNALING Endo . 1995 Vol 136 . No 10

FIG. 1. Displacement of [12511spGIP binding from COS-7 cells transiently expressing GIP-Rl (A) and the stable CHO-Kl cell line rGIP-15 (B-D). All are representative curves of three or more individual experiments. Different commercial preparations were tested (A and B): shGIP-1 and spGIP-1 (Bachem), and shGIP-2 and spGIP-2 (Peninsula). No significant differences (n 2 3) in the potencies of the different forms or preparations were found with either system. The ability of GIP fragments (C) and related hormones (D) to inhibit [lz511 spGIP was also tested. C, Of the fragments, only GIP-(l-30)-OH displaced [1251]spGIP binding significantly. D, The exendins, Ex-4 and Ex-(g-391, inhibited binding by 21% and 39%, respectively, at 1 P-“I. Other fragments and peptides tested that had no effect on binding included GIP-(17-301, GLP- l-(21-26), glucagon-(21-261, VIP, GLP-2, and glucagon (data not shown).

validating the use of more readily available synthetic preparations for use in GIP studies. Nontransfected CHO-Kl cells did not display specific [‘251]spGIP binding (data not shown).

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To localize the region of GIP required for receptor binding, the abilities of several GIP fragments [shGIP-(l-30)-OH, sp- GIP-(17-30), and spGIP-(19-30)] (reviewed in Ref. 2) to displace [7251]spGIP binding from rGIP-15 were examined. In addition, based on the recent identification of the region 21-29 as a biologically active fragment of glucagon (24), several fragments with sequences based on the homologous region 21-26 in GIP, tGLP-1, and glucagon were synthesized and tested. These included GIP-(21-261, GIP-(l&28), tGLP- l-(21-26), and glucagon-(21-26) [GLU-(21~26)]. As shown in Fig. lC, GIP-(I-30)-OH completely displaced [‘251]spGIP, but with lower affinity (IC,, = 39 +- 17 nM) than that for spGIP- (l-42) (IC,, for spGIP-2, 3.6 5 0.4 nM) None of the-other

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fragments was able to compete significantly with [‘251]spGIP binding at concentrations as high as 1 j&M (Fig. 1C).

The specificity of the receptor was examined further in rGIP-15 cells using several structurally related mammalian peptides [tGLP-1, GLP-2, glucagon, VIP, Ex-4, and a truncated form of exendin, Ex-(9-39); Fig. 1D and data not shown]. The venom peptides isolated from Heloderma sus- pectum, Ex-(9-39) and Ex-4, demonstrated significant low affinity binding to the GIP receptor, with approximately 39% and 21% displacement of [‘251]spGIP, respectively, at 1 PM

and with similar displacement seen in COS-7 cells transiently expressing GIP-Rl (data not shown). None of the other peptides tested had any effect on binding. Although these results demonstrated the specificity of the receptor for GIP, they also suggest that Ex-(9-39), an antagonist of the GLP-1 receptor, may have a similar action at the GIP receptor when used in the micromolar or higher concentration range.

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GIP RECEPTOR. BINDING AND SIGNALING 4633

Effects of rat islet GIP receptor expression on CAMP formation

To correlate GIP receptor binding to the activation of the adenylyl cyclase system, CAMP accumulation was determined by RIA in COS-7 cells expressing GIP-Rl. In the presence of IBMX, synthetic porcine GIP evoked a concentration- dependent increase in CAMP accumulation (EC,, = 8.7 +- 1.5 X 10-l’ M). This effect was not significantly different from that observed with shGIP (8.1 + 1.6 X 10-i’ M; Fig. 3), indicating that the human and porcine species of GIP share similar biological activities. In the absence of IBMX, a qual- itatively similar dose-response relationship was observed; however, maximal CAMP accumulation was approximately one fourth of the level measured in its presence (data not shown). No significant increases in CAMP accumulation were observed with any of the structurally related peptides tested previously in binding experiments, with the exception of Ex-4, to which a small response (2.6 2 0.3-fold over basal) was observed at the highest concentration tested (1 PM). In control experiments with cells expressing the GLP-1 receptor, GIP (100 nM) was unable to evoke a CAMP response, whereas a 5-fold increase in CAMP accumulation was observed with 1 nM tGLP-1 (data not shown), further demonstrating the specificity of the response in COS-7 cells.

The rGIP-15 cell line was used subsequently to examine the effects of different GIP preparations, related hormones, and fragments on GIP-stimulated activation of CAMP production (summarized in Table 1). Due to the availability of large number of cells expressing GIP-Rl, it was not necessary to include IBMX in this assay system, and the CAMP respon- siveness of rGIP-15 was similar to that seen in COS-7 cells transiently expressing GIP-Rl. Comparisons of CAMP responses to different synthetic GIP preparations did not re- veal any significant differences at any GIP concentration examined (Table 1). The preparations of shGIP used in the present study produced an identical insulin response to spGIP in the isolated perfused rat pancreas (integrated 50 min insulin responses: glucose alone, 105 + 22 nmol; shGIP-1,1036 2 175 nmol; spGIP, 1057 ? 196 nmol; shGIP-2, 1050 + 161 nmol; spGIP-2, 1085 ? 105 nmol; Table l), in contrast to commercial preparations of shGIP bioassayed in

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previous studies (16). As would be expected, given the binding data, GIP-(l-30)-OH was the only fragment that stimulated CAMP accumulation, and it was equipotent to spGIP-(l-42) at all but the lowest concentration tested (0.10 nM; P < 0.05; Table 1). In the isolated perfused pancreas, the integrated insulin responses to GIP-(l-30)-OH (861 t 95 nmol/50 min; Table 1) were not significantly different from those in response to the synthetic GIP preparations tested (Table 2). None of the other fragments potentiated or antag- onized 10 nM spGIP-stimulated CAMP accumulation (data not shown).

As the GLP-1 antagonist Ex-(9-39) and agonist Ex-4 dis- played binding to the rat pancreatic islet GIP receptor, the abilities of these peptides to alter CAMP accumulation in the absence and presence of GIP were examined. In rGIP-15, and in contrast to experiments with transiently transfected cells, Ex-4 did not increase CAMP measurably at concentrations as high as 1 PM, nor did it antagonize GIP-stimulated cAMP increases (Fig. 4A). Importantly, Ex-(9-39) did not have any effect on basal CAMP accumulation or its response to 10 nM spGIP (Fig. 4A). In agreement with our previous studies (251, both Ex-4 and Ex-(9-39) exerted their expected agonist and antagonist effects, respectively, in cells expressing the GLP-1 receptor (Fig. 4B). As binding data would predict, none of the related hormones [GLP-l-(7-36), GLP-2, glucagon, or VIP] had any effect on cAMI’ accumulation in rGIP-15 (data not shown).

The glucose-dependent nature of the GIP-induced insulin response is well documented. Although unlikely, it has not been clearly established whether the coupling of the receptor to the adenylyl cyclase system involves a glucose-dependent step. The availability of the cloned receptor has allowed this issue to be addressed. COS-7 cells transiently expressing GIP-Rl were preincubated in varying glucose concentrations for 1 h and tested under basal and GIP-stimulated conditions (10 nM) for 30 min (Table 2). Although both basal and GIP- stimulated CAMP accumulation were lower in 0.1 mM glucose-treated COS cells, GIP was able to evoke a large (22-fold) increase in CAMP accumulation. GIP had an equivalent effect on CAMP in the presence of glucose concentrations of 5.5 and 16 mM. These data suggest that under the experimental conditions used in this study, the glucose concentration had little effect on GIP-stimulated CAMP accumulation.

Effects of GIP on [Ca”+],

Usdin et al. (14) demonstrated that the RINm5F cell GIP receptor, when expressed in a calcium reporter cell line (HEK293 expressing apo-aequorin), yielded an increase in [Ca’+], in the presence of 100 nM GIP. The present series of experiments was designed to examine the linkage between GIP-Rl and [Ca2+li. In COS-7 cells expressing GIP-Rl and loaded with the Ca2+ indicator fura-2/AM, 50 nM GIP increased [Ca’+], with an acute transient phase, followed by a sustained elevation of [Ca2+li (Fig. 5A). The net increases in the transient (Pl; AlCa2+li) and sustained (P2; A[Ca2’li) phases were 114 -C 8.1 and 36 +- 6.1 nM, respectively (n 2 3). A[Ca*+], Pl was further shown to be concentration dependent, with net increases of 49 2 3.8 and 11 t 4.5 at 5 and 0.5 nM spGIP, respectively (n = 3). To determine whether the source of the increased [Ca*+]i elicited by GIP was intracel-




TABLE 1. The effects of different GIP preparations, and GIP-(l-30)-OH on CAMP accumulation (fold increase over basal) in rGIP-15 cells and insulin release from the isolated perfused rat pancreas (integrated insulin response, nanomoles over 50 min)

Treatment shGIP-1 spGIP-1

0.1 nM GIP 5.8 t 0.3 6.0 i 0.1 1 nM GIP 7.6 t- 1.0 7.8 ? 0.3 10 nM GIP 9.6 k 1.2 9.4 k 1.2 Integrated insulin 1036 ? 105 1057 ? 196

Data presented are the mean 2 SEM (n 2 3 independent observations).

shGIP-2 spGIP-2

5.7 + 0.6 6.5 k 0.1 6.6 2 0.5 8.2 ? 0.4 8.8 L 0.3 10.0 +- 2.5

1050 + 161 1085 t 105

GIP-(l-30)

4.9 t 0.3" 8.3 2 0.3 7.8 k 0.4

861 t 95

Q P c-0.05.

A

125 1

FIG. 4. The effects of Ex-4 and Ex-(9-39) on spGIP-stimulated CAMP formation in the stable CHO-Kl clone rGIP-15 (A), and tGLP-l-stimulated CAMP formation in cells stably expressing the rat GLP-1 receptor (B). Neither form of Ex influenced GIP-stimulated CAMP production (A), whereas Ex-4 increased CAMP levels, and Ex-(9-39) inhibited tGLP-l-stimulated CAMP production (**, P < 0.01; B).

TABLE 2. The effect of glucose concentration on spGIP- stimulated CAMP accumulation in COS-7 cells transiently expressing GIP-Rl

Treatment 0.1 mkx glucose 5.5 mid glucose 16 IWVI glucose (pmolhl) (pmoVml) (pmoliml)

Basal 2.8 k 0.1” 4.7 t- 0.4 6.5 t 0.6 spGIP (10 nM) 61.2 -f 0.6” 83.5 L 7.5 78.5 ? 3.6

Data presented are the mean ? SEM (n 2 3 independent observations).

"P < 0.05.

Mar or extracellular, the above experiment was repeated first in a nominally Ca ‘+-free environment (Table 3) and then in the presence of 4 mM EGTA (Fig. 5B). Under both conditions, the transient first phase response was reduced, but not eliminated (70 5 7.6 and 40 t 7.6 nM, respectively, ZIS. 114 ? 8.1 nM in controls; P 5 0.05). In contrast, the second phase

A. B. 400 -

GIP

f 300- +

250-

EGTA 200- 1 Glp

loo- 1, loo- II

, , , , , ,

1,

, , , , , ,

0 100 200 300 0 100 200 300

Time (set) Tlme (set)

FIG. 5. Effects of spGIP on [Ca2+li in suspensions of COS-7 cells. A, The effect of spGIP on [Ca2+li was measured in COS-7 cell suspensions 72 h posttransfection with pGIP-Rl. The cells were loaded with fura-2, and then spGIP (50 no) was added at time point indicated by the arrow. In control experiments, GIP was unable to evoke a [Ca”], response in cells expressing the GLP-1 receptor under identical conditions (not shown). B, To determine the source of the spGIP-induced increase in [C!a2+li, GIP-Rl-transfected COS-7 cells were preincubated in 4 mM EGTA and stimulated with 50 nM spGIP. Alternatively, the cells were pretreated with 10 yM nifedipine (C) or the Ca2+- ATPase inhibitor, thapsigargin (50 nM; D). Tracings are representative of at least three independent experiments.

responses were completely eliminated; in fact, spGIP ap- peared to induce Ca *+effluxfromthecell(-10 + 2.4and -17 t 2.4 nM). These data are consistent with the transient increase in [Ca2+li originating primarily from an intracellular Ca*+ pool and the sustained phase of the [Ca*+l, increase resulting from an extracellular source(s).

To characterize the first phase [Ca*+l, response further, cells were exposed to the sarcoplasmic/endoplasmic reticulum Ca*+ adenosine triphosphatase (ATPase) inhibitor, thapsigargin, in nominally Cazc-free medium. Thapsigargin (50 nM) initially caused an increase in [Ca*+l, followed by a plateau phase, suggesting depletion of intracellular Ca*+ stores. Subsequent addition of GIP (50 nM) did not elicit an increase in [Ca*+l. strongly suggesting that the Pl response was primarily due to the mobilization of Ca2+ from intracellular stores. To characterize the Ca*+ entry pathway, COS-7 cells expressing GIP-Rl were pretreated with the L- type VDCC blocker, nifedipine (10 FM; Fig. 5C). Nifedipine had no effect on the sustained increase in [Ca2+li (42 + 2.9 us. 36 ? 6.1 nM; P > 0.05) or on the immediate acute rise in [Ca2+], (110 2 5.3 ZIS. 114 t 8.1; P > 0.05; Table 3). This result is in contrast to our previous findings in insulin-secreting



GIP RECEPTOR, BINDING AND SIGNALING 4635

TABLE 3. Effects of spGIP on [Caztli in COS cells expressing GIP-Rl

Treatment cos-7 a [w+l ’

cos-7 n [C2’1, P1 (IIM) P2 bI)

50 nM GIP 114 * 8.1 36 2 6.1 50 nM GIP + 10 pM nifedipine 110 2 5.3 42 t 2.9 50 nM GIP (Ca’+-free medium) 70 k 7.6 -10 2 2.4 50 nM GIP + 4 mM EGTA 40 k 7.6 -17 i 2.4

Data presented are the mean ? SEM (n 2 3 independent observations). Pl, Peak transient phase; P2, plateau phase.

HIT cells (12) and more recently in the PTC6-F7 cell line (unpublished results), in which EGTA (4 mM) pretreatment or L-type Ca2+ channel blockers prevented spGIP-induced increases in [Ca*+],. The activation of voltage-sensitive Cazt channels by spGIP in COS cells was further discounted, because KCl, used at a concentration that should depolarize the cell (50 mM), was unable to stimulate Ca2+ entry. The I’2 response was also not elicited by forskolin (10 PM) or IBMX (data not shown), indicating that spGIP-induced increases in [Ca2’li were not regulated by a protein kinase A-mediated pathway.

Confocal microscopy was used to determine the relative number of COS-7 cells responding to spGIP, and to examine Ca2+ fluxes in individual cells. In cells loaded with fluo3/AM, the majority had similar resting Ca2+ fluorescence levels (Fig. 6B). In response to the addition of 50 nM GIP, approximately lo-60% of the cells in any given field showed an increase in Cazf fluorescence intensity (Fig. 6C). This percentage is similar to that observed in control transfection experiments using pCMV P-galactosidase (In- vitrogen) as a reporter system to assess transfection effi-

ciency. The changes in fluorescence for each cell (indicated by UYYOZUS in Fig. 6B) were normalized to respective control values and plotted against time (Fig. 6D). The pattern of fluorescence, although similar among cells, varied greatly in overall intensity. When averaged, however, the calcium response pattern was remarkably similar to that observed by fluorimetry (Fig. 6D): that is, a rapid initial phase followed by a sustained second phase.

Discussion

The pancreatic islet GIP receptor has proven difficult to study due to its low level of expression and difficulties in obtaining islets in sufficient quantities for detailed investi- gations. In light of these limitations, much of the information regarding GIP receptor expression has been obtained from membranes of multipotential islet-derived tumor cells that express a variety of neuroendocrine peptide receptors (reviewed in Ref. 2). The isolation of a cDNA encoding a single species of the rat islet GIP receptor has provided us with a tool to specifically assess the ligand binding and intracellular signaling properties of this protein, studies that are clearly important given the therapeutic potential of the incretins for the treatment of NIDDM. Specifically, receptor cDNAs (GIP- Rl and GIPR2) have been isolated that are homologous to that obtained by PCR from the rat tumor cell line (RINm5F) (14), establishing that this receptor is present in pancreatic islet cells. Usdin et al. (14) detected increases in CAMP and [Ca2+], using reporter cell lines. In the present study, direct binding of the ligand to the receptor was demonstrated, and the ability of a number of other ligands and fragments to

FIG. 6. Effect of spGIP on [Caz+l, in individual COS-7 cells. To determine the pattern of the changes in [Ca?,, confocal microscopy was employed. A, Non- confocal image acquired using the Bio- Rad transmitted light attachment showing COS-7 cells transfected with pGIP-Rl. Scale bar = 50 pm. B, Con- focal image of the same cells as in A loaded with fluo-S/AM, showing resting Ca2+ levels. Relative fluorescence ap- pears in color scale, with blue representing lower and red representing higher Ca2+ fluorescence. C, In response to 50 nM spGIP, fluorescence was followed in five identified responding cells (no. l-5). D, Changes in Ca2+ fluorescence, normalized to the resting fluorescence (%AF/F) for each cell, were analyzed over time from the identified cells (no. l-5). GIP was added at time zero. E, The Caa+ signals for cells l-5 were averaged to show the Caa+ signal in a population.

D) Ca” signals, identified cells 1.50 r

E) Total Ca*’ signal 150 r

ii 125-

5

s o IOO-

i! 75-

.b : cn 50: r:

0” 257

o-

1. I I I I I

0 100 200 300 400

Time (set)

[.I,,., I,.,, 1 ,,.I., .I..

0 100 200 300 400

Time (set)




interact with the receptor were investigated. Increases in CAMP in response to GIP and a number of other peptides were directly measured using a specific RIA. A more sensitive form of s ectrofluorimetry allowed real time measurements of [Ca E Ii increases in response to GIP and characterization of the Cazt sources involved in the response.

Cells transiently and stably expressing GIP-Rl and GIP-R2 demonstrated a single class of high affinity binding sites [spGIP-2 IC,,, 3.6 5 0.4 nM, which is within the range of those reported in tumor cell lines (reviewed in Ref. 2) and that of a receptor cDNA recently cloned from the hamster p-cell line, HIT-T15 (26)]. With both intact CHO-Kl cells and membranes, binding isotherms were monophasic, with apparent Kd values of 200-300 PM. In addition, synthetic porcine GIP yielded concentration-dependent increases in CAMP, with an EC,, of 8.7 X 10-l’ M, a value in agreement with those previously reported in HIT cells (12,26; reviewed in Ref. 2) and that correlates well with the apparent K, (-200 PM). The observation that GIP-(l-42) and GIP-(l-30) were the only peptides examined, including several other members of the secretin/VIP family, that displaced [‘251]spGlP-2 and stimulated CAMP accumulation in the nanomolar concentration range reinforces the proposal that the cDNA isolated from RlNmF5 cells (14) and here from purified pancreatic islets encodes a GIP-specific receptor. One predicted amino acid difference between the GIP-R1 and RINm5F receptor (Gluz1-+Gln2*) (14) was observed in the two independent cDNAs analyzed. This polymorphism may be explained by differences in the strain of the rodents used to obtain the RNA or may represent a PCR-based mutation. It is difficult to assess the significance of this substitution at this time because ligand binding data are not currently available for the RINm5F receptor.

Although GIP has potent insulinotropic properties and facilitates glucose disposal, controversy exists as to the relative effectiveness of GIP 7~s. tGLP-1 in stimulating insulin release. Some investigators have found GIP to be equipotent to tGLP-1(27,28), whereas others have found that tGLP-1 has markedly greater insulinotropic activity (29,30). In addition, GIP was shown to be strongly insulinotropic in humans in one study (4), whereas a subsequent report by the same researchers demonstrated that GIP was a poor stimulant of insulin secretion (7). Some of these differences may be explained by variability in the relative potencies among several commercially available GIP preparations. Having a single species of the rat GIP receptor expressed in COS-7 and CHO-Kl cells has provided us with an ideal model with which to begin to address this issue. The present studies revealed no significant differences between the porcine and human GIP formulations tested with respect to binding properties or the ability to stimulate CAMP accumulation and suggest that the rat GIP receptor does not have preferential affinity for either peptide. These findings indicate that the His”/Arg” and Ser34/Asn34 amino acid differences between porcine and human GIPs do not influence ligand- receptor interaction. These studies also revealed no significant difference in the affinities or potencies of synthetic GIP preparations obtained from two different suppliers. Unlike earlier peptide preparations (15), the current batches of spGIP and shGIP were equipotent as insulinotropic agents in

the isolated perfused rat pancreas. This indicates that porcine and human GIP bind to and activate the rat islet GIP receptor equally and implies that such a heterologous system can be used for structure-activity studies on human and porcine GIP. Also, although the present studies cannot conclusively confirm that the discrepancies seen in the literature concern- ing the insulinotropic potency of shGIP are due to variations in the preparations, they do indicate that GIP-Rl-expressing cell lines represent an ideal model to assess such preparations in the future.

In humans, GIP has been shown to be a potent incretin (l-3), and because incretins and their analogs have promis- ing therapeutic potential for the treatment of NIDDM (7,8), it is important to develop an understanding of the regional sequence requirements for receptor interaction. Previous studies by ourselves and others have localized the biologically active region of the molecule to residues 15-30 (reviewed in Ref. 2). GIP-(l-30)-OH displaced [‘251]spGIP binding with an affinity -12-fold less than that of GIP-(1-42) (IC,,, 39 nM; spGIP-2 IC,,, 3.6 nM; Fig. lC), but was equipotent to the intact molecule in stimulating CAMP accumulation at all but one concentration examined (0.10 nM). GIP- (l-30)-OH yielded an integrated insulin response not significantly different from that of GIP-(1-42) [GIP-(l-30), 861 -C 95 nM/50 min; shGIP-2,1085 + 105 nM/50 min). These data indicate that this peptide shares the same site of action as the native peptide, but that the missing carboxy-terminal residues may contribute to receptor-ligand interactions.

To define the biologically active region further and to examine the possibility that a common biological core exists among GIP, tGLP-1, and glucagon that could be exploited for the possible design of antagonists and agonists, we examined the abilities of a number of peptide fragments to bind to the GIP receptor and stimulate or reduce CAMP production in rGIP-15 cells. None of the peptides tested (based on the semiconserved region of amino acids 21-28 of GIP, tGLP-1, and glucagon) exhibited any ability to inhibit binding. Sur- prisingly, GIP fragments 17-30 and 19-30 had no effect on [1251]spGIP binding or CAMP accumulation despite having been demonstrated to exert some insulinotropic activity in the perfused pancreas (our unpublished results). This sug- gests that they may be acting via interaction with either another related receptor or an unidentified isoform of the GIP receptor. This latter concept is supported by studies demonstrating multiple receptor subtypes for other peptide hormone receptors (31, 32). Alternatively, it may be that glycosylation plays a role in receptor function (33). Differ- ences in glycosylation in pancreatic p-cells compared with CHO-Kl cells may alter the ability of smaller fragments to bind and/or activate the receptor (33).

The use of the GLP-1 receptor antagonist Ex-(9-39) has recently provided information supporting an important role for tGLP-1 in the enteroinsular axis (34, 35). In our studies, Ex-4 and Ex-(9-39) were the only non-GIP peptides that displaced [‘251]spGIP binding, suggesting that at high concentrations, Ex-(9 -39) may act as a GIP antagonist. Even at 1 PM, however, Ex-(9-39) was unable to inhibit GIP-stimulated CAMP production, whereas in parallel studies with cells expressing the rat tGLP-1 receptor Ex-(9-39) inhibited GLP-l-stimulated CAMP production by approximately 65%.



GIP RECEPTOR, BINDING AND SIGNALING

These data support recent studies in rats (34,35) showing that Ex-(9-39) does not alter the contribution of GIP to the enteroinsular axis in uizm or decrease its insulinotropic action in RIN5AH cells (35) at the concentrations used. It is possible that the exendins have affinity only for the G-protein-un- coupled state of the receptor (20). However, we were unable to consistently distinguish a higher affinity state in saturation and competition assays using both intact cells and membrane preparations (data not shown). Efforts are currently underway in our laboratory to obtain a constitutively active mutant of the GIP receptor that will expedite the characterization of such a state and provide a more sensitive reporter system for the screening of putative receptor agonists and antagonists.

The major advantage of the incretins over the sulfonyl- ureas as potential therapeutic agents to treat NIDDM is that their actions are dependent on the ambient glucose concentration. The insulinotropic action of GIP is glucose dependent in viva (l-3), and a similar glucose dependence is observed on CAMP accumulation in cell lines (12). Similarly, the stimulation of CAMP production by the related peptide tGLP-1 in some cell lines (12) and in isolated p-cells (36) was also shown to be glucose dependent, whereas other studies have reported a lack of glucose dependence in some cell lines and transfected cells (37). As was expected, COS-7 cells transiently expressing the rat islet receptor exhibited GIP-stimulated CAMP accumulation even under nominally glucose- free conditions. This lack of glucose dependence for CAMP production demonstrates that a fundamental difference exists in G protein-linked regulation of CAMP levels in glucose-responsive p-cells and cell lines.

Usdin and colleagues (14) demonstrated that the GIP receptor, when expressed in a calcium reporter cell line (HEK293 cells expressing apo-aequorin), yielded an increase in [Ca’+l, in the presence of 100 nM GIP. However, the potential source(s) of the increased [Ca”], was not charac- terized. In the present study, COS cells expressing the GIP receptor were analyzed for changes in GIP-induced [Ca2-cli. When GIP (50 nM) was added to the GIP-Rl-transfected COS cells, [Caztli first rose rapidly to a peak (114 2 8.1 nM), termed Pl, and then decreased to a sustained level over basal (36 +- 17 nM), termed P2. Thus, the response was biphasic in nature. The sustained phase, P2, was abolished under conditions in which extracellular calcium was removed from the medium, suggesting that this response was dependent on Ca2+ influx across the plasma membrane. In contrast, the peak transient phase, Pl, persisted under Ca’+-free conditions, suggesting that it was dependent on the release of Ca2+ from intracellular sources.

To examine this possibility further, cells were treated with the Ca2+-ATPase inhibitor, thapsigargin. As thapsigargin is known to deplete the intracellular inositol trisphosphate- sensitive Ca2+ stores (38), the loss of the peak transient phase after this treatment lends supports to the possibility that Pl represents a GIP-induced mobilization of Ca” from intracellular stores. Although the ability of the endogenous GIP receptor to couple to Ca2+ through inositol trisphosphate has not been described in pancreatic B cells previously, the second incretin, tGLP-1, in part stimulates [Ca2+li through the release of intracellular Ca2+ in mouse p-cells (37), and we have shown previously that the recombinant tGLP-1 recep-

tor, when expressed in COS cells, couples to phospholipase C in addition to adenylyl cyclase (18). Although it is possible that the ability of the GIP receptor to activate intracellular Ca2+ stores may simply reflect an artificial situation resulting from expression of the GIP receptor in COS cells, other members of the secretin/VIP receptor gene family, including receptors for PTH/PTH-related pepide (39), glucagon (40), and calcitonin (41, 42), are capable of mobilizing intracellular Cazt stores. Given the apparently wide distribution of the GIP receptor in extrapancreatic tissues, it is possible that, as with the receptors for glucagon and pituitary adenylate cyclase-activating polypeptide, the GIP receptor couples to different G-protein species and signal transduction path- ways in different tissues (43, 44).

In previous studies using the glucose-responsive HIT-T15 cell line as a model, we demonstrated that GIP increased CAMP levels and promoted Ca2+ influx through VDCC, resulting in stimulation of insulin secretion (12). These studies, and others focusing on tGLP-1, suggested that both incretins target either the ATP-sensitive Kt channel and/or VDCC to increase [Ca2’], through CAMP-dependent protein kinases (12, 35, 45-47). Our findings that GIP evoked a concentration-dependent increase in CAMP accumulation in COS cells supports this model. However, as these cells do not bind dihydropyridines (our unpublished results) or respond to KC1 or forskolin, it is unlikely that the GIP-induced increase in Ca2+ influx was mediated by a CAMP-dependent process. This possibility is supported by the observation that the increase in [Ca’+], during the sustained phase (P2) was not sensitive to the L-type calcium channel antagonist nifedipine. As I’2 was dependent on extracellular Ca’+, GIP may activate a voltage-independent Ca2+/cation channel. Recent studies using the mouse insulin-secreting tumor cell line PTC6, demonstrated that a component of the tGLP-l-induced increase in [Ca2+li is not sensitive to nifedipine or membrane depo- larization (48). Holz and co-workers (48) suggested that in addition to its actions on ATP-sensitive Kf channels, tGLP-1 modulates P-cell Ca2+ . Influx through voltage-independent Ca2+ channels or nonspecific cation channels. Whether the voltage-independent Ca2+ influx evoked by GIP in COS-7 cells and that evoked by tGLP-1 in /3TC6 cells represent the same receptor-mediated Ca2+ entry pathway remains to be determined. Also undetermined is the significance of the heterogeneity in the Ca*+ flux patterns observed in individual cells. It is possible that the magnitude of the Ca2+ response is dependent on the level of receptor expression, representing another potential mode of agonist regulation. This relationship has been documented for the PTH/PTH- related peptide (49) and the calcitonin receptor isoforms Cla and Clb; an increase in the Ca2+ sensitivity to calcitonin was positively correlated with receptor number (41). Studies are now underway to explore the possible relationship between GIP receptor expression level and ]Ca’+l,.

Whether aberrations in the enteroinsular axis contribute to NIDDM remains unresolved. Recent genetic linkage studies suggest that mutations of the GLP-1 receptor are not causally related to this disease (50). Studies demonstrating that GIP is not effective compared to tGLP-1 in stimulating insulin secretion in NIDDM could be explained by a greater potency of tGLP-1 over GIP for stim-



GIP RECEPTOR, BINDING AND SIGNALING Endo . 1995 Vol 136 . No 10

ulating insulin secretion in this disease. Conversely, it may suggest a linkage between NIDDM and the GIP receptor. The cloned rat islet receptor should prove useful for the subsequent isolation of the human isoform for genetic linkage studies and further aid in the development of GIP analogs with greater therapeutic potential.

Acknowledgments

The authors would like to thank Milton P. Charlton (Department of Physiology, University of Toronto) for Ca ‘+ imaging and analysis. The confocal facility was provided by the Neuroscience Network. We would also like to thank Gregory P. Downey (Department of Medicine, Uni- versity of Toronto) for the use of fluorimetry equipment and for technical assistance with Ca2+ measurements, and Iibss-T.-A. McGillivary, H&me C&?. and Ieff Hewitt (Deuartment of Biochemistrv, Universitv of British Colimbiaj for help and a&ice on molecular bioldgy, tissue &lture, and transfection techniques.

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