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Structural requirements for gastric inhibitory polypeptide (GIP) receptor binding and stimulation of insulin release
Peptide,~,
Vol. 7, Suppl. 1, pp. 75-78, 1986. <' Ankho International Inc. Printed in the U.S.A.
0196-9781/86 $3.00 + .00
Structural Requirements for Gastric Inhibitory Polypeptide (GIP) Receptor Binding and Stimulation of Insulin Release MAURA MALETTI,*' MATS CARLQUIST,t BERNARD PORTHA,$ MICHELINE KERGOAT,~ VIKTOR MUTTt AND GABRIEL ROSSELIN§
*Fondation de Recherche en Hormonologie, 94260 Fresnes, France tDepartment o f Biochemistry II, Karolinska lnstitutet, S-104 Ol Stockholm, Sweden SLaboratoire de Physiologie du D#veloppement, C N R S LA-307 Universit# Paris VII 75251 Paris Cedex 05 France §Unit# I N S E R M U55, HOpital Saint-Antoine, 75571 Paris Cedex 12, France
MALETTI, M., M. CARLQUIST, B. PORTHA, M. KERGOAT, V. MUTT AND G. ROSSELIN. Structural requirements fi)r gastric inhibitory polypeptide (GIP) receptor binding and stimulation of insulin release. PEPTIDES 7: Suppl. 1, 75-78, 1986.--The effect of bovine GIP 1-42 and several of its fragments in competing with thebinding of ~2:'I-GIPto /3-cell plasma membranes from transplantable hamster insulinoma, and in stimulating insulin release from the isolated perfused rat pancreas, was investigated. Our results, in association with the results of previous studies, indicate that the sequence 17-38 is necessary for receptor binding and biological activity of GIP. By contrast, the N-terminal portion of GIP can be removed without seriously impairing the activity of the molecule. Bovine GIP
GIP receptor
/3-Celltumor
Biological activity
been described [3]. Briefly, a glutamic acid specific enzyme (Staphylococcus aureus V8) cleaved off the N-terminal tripeptide from GIP, resulting in GIP 4-42. Cleavage with enterokinase gave two main fragments, 1-16 and 17-42. Further cleavage of the fragment 17-42 with trypsin resulted in GIP 1%30.
HIGH affinity and specific binding sites for the porcine gastric inhibitory polypeptide (pGIP) have been found [7] and characterized [8] in the plasma membranes of r-cells from transplantable hamster insulinoma. Vasoactive intestinal peptide (VIP), glucagon, secretin, peptide having N-terminal histidine and C-terminal isoleucine amide (PHI), all of porcine origin and human pancreatic growth-hormone releasing factor (hpGRF), which share with pGIP 4, 15, 9, 7 and 5 identities respectively, do not exhibit any effect on the GIP receptor [7,8]. The aim of this work is to investigate whether the binding and the biological activity of GIP are due to a part of the molecule or to the whole structure by using the whole molecule and different fragments of bovine GIP (bGIP) [3]. bGIP differs from pGIP by an isoleucine residue at position 37 instead of lysine. The ability of those molecules to inhibit the binding of the 125I-ligand to/3-cell plasma membranes and to stimulate insulin release from the isolated perfused rat pancreas was measured.
Preparation of Radiolabeled Peptides The HPLC-purified pGIP 1-42 and, in some experiments, bGIP 4-42 were radiolabeled with Na '"5I by the chloramine-T procedure to 50-80 /~Ci//zg, i.e., 0.11-0.19 iodine atoms per molecule, to minimize the production of diiodinated derivatives. The iodinated material was diluted to 1 ml with 1% trifluoracetic acid (TFA) buffered to pH 2.5 with diethylamine (DEA) and immediately applied to a SepPak C18 cartridge [9]. The eluted '2~l-labeled GIP was purified by HPLC on a/x-Bondapak C18 column, as previously described [8].
METHOD
Insulin Releasing System
bGIP and Fragments
The biological activity of bGIP 1-42, 17-42 and 4-42 was tested according to their ability to stimulate insulin release from the isolated perfused rat pancreas. Isolation and perfu-
The procedure for purification of bGIP and the fragmentation and identification of peptide fragments has recently 'Requests for reprints should be addressed to Maura Maletti.
75
M A L E T T I ET AL.
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FIG. 1. Competitive inhibition of ~I-GIP 1-42 binding by unlabeled bG1P 1-42 and its fragments 4-42, 17-42, 1-16, 1%30 and 1-3. Binding assay conditions were described in details elsewhere [8]. Each point is the mean of duplicate determinations in three separate experiments. -//
116
126
FRACTION
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FIG. 2. Purification of ~2~I-bGIP 4-42 by HPLC. The iodination mixture resulting from the Sep-Pak C18 purification was applied to a /z-Bondapak C18 column and eluted with a gradient of CH:~CN in lC~ TFA pH 2.5.
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FIG. 3. Competitive inhibition of "-"~I-bGIP 4-42 binding by unlabeled bGIP 1-42 and its fragments 4-42, 17-42, 1-16 and 1%30. Other details as in Fig. 1.
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sion o f the rat p a n c r e a s w e r e p e r f o r m e d as p r e v i o u s l y described [4].
Preparation of Membranes M e m b r a n e s w e r e p r e p a r e d from insulin-secreting h a m s t e r p a n c r e a t i c t u m o r s . The t u m o r s [61 w e r e serially t r a n s p l a n t e d s u b c u t a n e o u s l y in Syrian h a m s t e r s [5,6]. A n i m a l s b e a r i n g
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FIG. 4. Effect of 5 ng/ml bGIP 1-42 and 17-42 on the insulin response to 6.6 mM glucose by the isolated perfused rat pancreas. Insulin response is expressed as the stimulated insulin release to basal insulin release ratio. Data are the mean of two separate experiments. The effect of 19 mM arginine (ARG) was taken as a control response.
S T R U C T U R E - F U N C T I O N R E L A T I O N S H I P OF GIP
77 RESULTS
100
Competitive Inhibition of '2"~I-GIP 1-42 by GIP Fragments
,17-42
The effect o f b G I P 1-42 and its fragments 1-3, 4-42, 1-16, 17-42 and 1%30 in competing with the binding of '2'~I-GIP 1-42 to the transplantable hamster insulinoma is shown in Fig. 1. GIP 1-42, 4-42 and 17-42 competitively inhibited the binding of the tracer. Half maximal inhibition of ~z'~I-GIPwas obtained at 6.6×10 ~ M, 2.2× 10 9 M and 10-6 M with GIP 1-42, 4-42 and 17-42 respectively. Fragments 1-3, 1-16 and 1%30 have no competitive effect at a concentration of 10-'~ M.
Purification of '2~I-GIP 4--42 To further investigate the similarity of bG1P 1-42 and 4-42 in interacting with the GIP receptor, we labeled bGIP 4-42 with ~Z~lto trace the reaction. '2'~I-labeled GIP 4-42 was separated in two main peaks by HPLC as shown in Fig. 2. A preferential binding tc plasma membrane of the transplantable hamster insulinomawas found with peak I of '25I-GIP 4-42, which was used for further experiments.
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, 11
The results are shown in Fig. 3. The pattern of the curves is similar to that obtained when'2'~I-GIP 1-42 was used as a tracer. Half-maximal inhibition of '2:'I-GIP was obtained at 5× 10-' M, 5× 10 " M and 5× 10-7 M with GIP 1-42 4--42 and 17-42 respectively. GIP 1-16 and 1%30 (10 5 M) were also ineffective on the binding of '~I-GIP 4-42.
[lIIIII
Biological Activity of GIP 17--42 and 4--42
,,,,,,,,
10-e
10-7
10s
10-s
GIP CONCENTRATION (M) FIG. 5. Comparison of bovine and porcine GIP by competitive inhibition of '2'~I-pGIPby unlabeled GIP 1-42 and its fragments 4-42 and 17-42. The stripped area represents the competition due to bGIP (mean+SEM). The inhibition curves of pGIP 1-42, 4-42 and 17-42 are indicated by: • and • respectively.
the tumor were killed approximately 60 days after grafting. The tumors were dissected, homogenized in 4 vol solution of 0.25 M sucrose, 0.01 M triethanolamine, 5 mM EDTA, pH 7.5 with a Dounce homogenizer, and centrifuged at 2,000 × g for 20 min. The supernatant was centrifuged at 20,000 × g for 20 rain; the resulting pellet was suspended in 6 mM HEPES, pH 7.5, and centrifuged at 20,000 × g for 20 min. Membrane pellets were stored at -80°C until use. Protein levels were determined by the method of Bradford [1].
Binding Assay The binding assay technique used here was essentially the same as that reported previously [8]. Briefly, membranes (0.5 mg protein/ml) were incubated with a fixed quantity of '251-GIP (about 1× 10-" M) and increasing amounts of natural GIP. The buffer was 5 mM MnCI2 in 60 mM HEPES, pH 7.5, containing 3% (w/v) BSA and 1 mg/ml bacitracin. After a 60-min incubation at 10°C, bound '25I-GIP was isolated by centrifugation and the radioactivity of the washed pellet was determined with a gamma counter. In all experiments, data are reported as specific binding by subtracting the nonspecific binding from the total, i.e., the amount of labeled peptide that was not displaced by an excess of natural GIP (10 '~ M).
As the results do not demonstrate if GIP 17-42, bound to the receptor sites, acts as an agonist or an antagonist of GIP, we have studied its effect in stimulating insulin release from the isolated perfused rat pancreas, bGIP 1-42 and 17-42 were infused for 15 min to achieve a perfusate concentration of 5 ng/ml in the presence of 6.6 mM glucose, according to the procedure previously described to test GIP fragments [13]. Both peptides produced an increase in insulin release (Fig. 4). The effect of the GIP 17-42 fragment corresponded to 32% of GIP 1-42 activity. This indicates that the 17-42 fragment is not an antagonist of GIP in this system. Infusion of bGIP 4--42 in six rat pancreas, under the same conditions, resulted in a small stimulation of insulin release as compared to bGIP 1-42 in only half of them. The biological activity of bGIP 1-16 and 1%30 was not tested, because of the complete loss of binding property found with these fragments (Fig. 1) and the loss of insulinotropic activity previously observed with pGIP 1-14 [13].
Comparison of Bovine and Porcine GIP by Binding Experiments Bovine and porcine GIP were compared using '2~I-pGIP as a tracer. As shown in Fig. 5, no difference was observed between the porcine and bovine GIP 1-42, 4-42, and 17-42, suggesting that the residue which is different (Ile-37 instead of Lys-37) does not induce any modification of the binding of GIP to the receptor. DISCUSSION
The comparison of the biological activity of GIP with its fragments has not yet been carried out because of difficulties in obtaining the fragments in sufficient amounts for a complete dose effect.
78
MALETTI E T A L .
In the results presented here two different systems have been used to characterize the GIP binding sites and the biological activity of GIP. Our data indicate that GIP 17-42 retains the property to bind the GIP receptor and, at least partly, to stimulate insulin release. It should be noted that high doses of GIP 17-42 are needed for competing with v-"~IGIP, whereas an insulinotropic activity of the fragment is observed at doses as low as 5 ng/ml. This suggests that even a low affinity binding of the ligand to the receptor could ensure a biological effect. Previous study on the effect of pGIP fragments on insulin release has shown that the C-terminal GIP 15-43 at a dose level of 10 ng/ml is about 40% as potent as intact GIP at 5 ng/ml, in the presence of 8.9 mM glucose [13]. The present results demonstrate that the activity of GIP 4-42, when observed, is slight, corresponding to approximately 10% of that of GIP 1-42. The absence of any insulinreleasing effect was noticed previously for pGIP 3-42, when infused in rat at a concentration of 5 ng/ml, in the presence of 8.8 mM glucose [2,10]. Our data suggest that the binding properties of GIP 4-42 are not directly related to its ability to stimulate insulin release. Indeed GIP 4-42, which competes as well as GIP 1-42 with '2~I-GIP binding, produces a small increase in insulin release, if any. In this regard, fragment 4-42 behaves like a partial antagonist of GIP. By contrast, it is of interest that suppression of the C-terminal part of pGIP as in GIP 1-38 did not result in any loss of the insulinotropic activity [12]. Shortening GIP to a
1%30 fragment suppresses the binding property of the molecule. Similarly, no effect was found with GIP 1-16 fragment. This result is supported by the complete loss of insulinotropic activity previously observed with pGIP 1-14 [131. Therefore the shortest GIP fragment showing both binding and insulinotropic activity is the 17-42 fragment. The present results, together with the effect of GIP 1-38 in stimulating insulin release [12], stress that the structural requirements for the binding and biological activity of the molecule are contained within the sequence 17-38. Interesting, however, is the observation that the differences between the porcine, bovine and human [11] GIP molecules are located in this 17-38 sequence, in positions 37 (bovine) or 18 and 34 (human). Our present findings also suggest that the N-terminal portion of GIP can be removed without impairing the activity of the molecule. Further studies, using G | P modified in the C-terminal part, are required to get further insight into the structurefunction of the interaction of GIP with its receptor. ACKNOWLEDGEMENTS This work was supported by grants from the Swedish Medical Research Council (project No. 01010) and the Nordisk lnsulinfond. We wish to thank Drs. J. Grenier, K. Nahoul and M. Roger for critical review of the manuscript, D. Bailbe, G. Cattoire and R. Victor Rapharl for their excellent technical assistance.
REFERENCES 1. Bradford, M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254, 1976. 2. Brown, J. C., M. Dahl, S. Kwauk, C. H. S. Mclntosh, M. Muller, S. C. Otte and R. A. Pederson. Properties and actions of GIP. In: Gut Hormones, 2nd edition, edited by S. R. Bloom and J. M. Polak. Edinburgh: Churchill Livingstone, 1981, pp. 248255. 3. Carlquist, M., M. Maletti, H. Jrrnvall and V. Mutt. A novel form of gastric inhibitory polypeptide (GIP) isolated from bovine intestine using a radioreceptor assay. Eur J Bioehem 145: 573-577, 1984. 4. Giroix, M. H., B. Portha, M. Kergoat, D. Bailbe and L. Picon. Glucose insensitivity and amino-acid hypersensitivity of insulin release in rats with non-insulin-dependent diabetes: A study with the perfused pancreas. Diabetes 32: 445-451, 1983. 5. Grillo, T. A. I., A. J. Whitty, H. Kirkman, P. P. Foa, S. D. Kobernick and R. Green. Biological properties of a transplantable islet cell tumor of the golden hamster. 1. Histology and histochemistry. Diabetes 16: 40%414, 1967. 6. Kirkman, H. A. A preliminary report concerning tumors observed in Syrian hamsters. Stanford Med Bull 20:163-165, 1962. 7. Maletti, M., B. Amiranoff, M. Laburthe and G. Rosselin. Mise en 6vidence de rrcepteurs sprcifiques du Peptide lnhibiteur Gastrique (GIP). C R Acad Sci [D] (Paris) 297: 563-565, 1983.
8. Maletti, M., B. Portha, M. Carlquist, M. Kergoat, M. Laburthe, J. C. Marie and G. Rosselin. Evidence for and characterization of specific high affinity binding sites for the gastric inhibitory polypeptide in pancreatic r-cells. Endoerinalo~,y 115: 13241331, 1984. 9. Marie, J. C., C. Boissard and G. Rosselin. Purification of monoiodinated vasointestinal peptide (MIZ~I-VIP)by high pressure liquid chromatography (HPLC). Peptides 5: 17%182, 1984. 10. Moody, A. J., K. Damm J0rgensen and L. Thim. Structurefunction relationships in porcine GIPDiabetolozia 21: 306, 1981 (abstr). 11. Moody, A. J., L. Thim and 1. Valverde. The isolation and sequencing of human gastric inhibitory peptide (GIP). FEBS Lett 172: 142-148, 1984. 12. Moroder, L., A. Hallet, P. Thamm, L. Wilschowitz, J. C. Brown and E. WOnsch. Studies on gastric inhibitory polypeptide: Synthesis of the octatriacontapeptide GIP 1-38 with full insulinotropic activity. Seand J Gastroenterol 13: Suppl 49, 129, 1978. 13. Pederson, R. A. and J. C. Brown. The insulinotropic action of gastric inhibitory polypeptide in the perfused isolated rat pancreas. Endocrinology 99: 780-785, 1976.
Vol. 7, Suppl. 1, pp. 75-78, 1986. <' Ankho International Inc. Printed in the U.S.A.
0196-9781/86 $3.00 + .00
Structural Requirements for Gastric Inhibitory Polypeptide (GIP) Receptor Binding and Stimulation of Insulin Release MAURA MALETTI,*' MATS CARLQUIST,t BERNARD PORTHA,$ MICHELINE KERGOAT,~ VIKTOR MUTTt AND GABRIEL ROSSELIN§
*Fondation de Recherche en Hormonologie, 94260 Fresnes, France tDepartment o f Biochemistry II, Karolinska lnstitutet, S-104 Ol Stockholm, Sweden SLaboratoire de Physiologie du D#veloppement, C N R S LA-307 Universit# Paris VII 75251 Paris Cedex 05 France §Unit# I N S E R M U55, HOpital Saint-Antoine, 75571 Paris Cedex 12, France
MALETTI, M., M. CARLQUIST, B. PORTHA, M. KERGOAT, V. MUTT AND G. ROSSELIN. Structural requirements fi)r gastric inhibitory polypeptide (GIP) receptor binding and stimulation of insulin release. PEPTIDES 7: Suppl. 1, 75-78, 1986.--The effect of bovine GIP 1-42 and several of its fragments in competing with thebinding of ~2:'I-GIPto /3-cell plasma membranes from transplantable hamster insulinoma, and in stimulating insulin release from the isolated perfused rat pancreas, was investigated. Our results, in association with the results of previous studies, indicate that the sequence 17-38 is necessary for receptor binding and biological activity of GIP. By contrast, the N-terminal portion of GIP can be removed without seriously impairing the activity of the molecule. Bovine GIP
GIP receptor
/3-Celltumor
Biological activity
been described [3]. Briefly, a glutamic acid specific enzyme (Staphylococcus aureus V8) cleaved off the N-terminal tripeptide from GIP, resulting in GIP 4-42. Cleavage with enterokinase gave two main fragments, 1-16 and 17-42. Further cleavage of the fragment 17-42 with trypsin resulted in GIP 1%30.
HIGH affinity and specific binding sites for the porcine gastric inhibitory polypeptide (pGIP) have been found [7] and characterized [8] in the plasma membranes of r-cells from transplantable hamster insulinoma. Vasoactive intestinal peptide (VIP), glucagon, secretin, peptide having N-terminal histidine and C-terminal isoleucine amide (PHI), all of porcine origin and human pancreatic growth-hormone releasing factor (hpGRF), which share with pGIP 4, 15, 9, 7 and 5 identities respectively, do not exhibit any effect on the GIP receptor [7,8]. The aim of this work is to investigate whether the binding and the biological activity of GIP are due to a part of the molecule or to the whole structure by using the whole molecule and different fragments of bovine GIP (bGIP) [3]. bGIP differs from pGIP by an isoleucine residue at position 37 instead of lysine. The ability of those molecules to inhibit the binding of the 125I-ligand to/3-cell plasma membranes and to stimulate insulin release from the isolated perfused rat pancreas was measured.
Preparation of Radiolabeled Peptides The HPLC-purified pGIP 1-42 and, in some experiments, bGIP 4-42 were radiolabeled with Na '"5I by the chloramine-T procedure to 50-80 /~Ci//zg, i.e., 0.11-0.19 iodine atoms per molecule, to minimize the production of diiodinated derivatives. The iodinated material was diluted to 1 ml with 1% trifluoracetic acid (TFA) buffered to pH 2.5 with diethylamine (DEA) and immediately applied to a SepPak C18 cartridge [9]. The eluted '2~l-labeled GIP was purified by HPLC on a/x-Bondapak C18 column, as previously described [8].
METHOD
Insulin Releasing System
bGIP and Fragments
The biological activity of bGIP 1-42, 17-42 and 4-42 was tested according to their ability to stimulate insulin release from the isolated perfused rat pancreas. Isolation and perfu-
The procedure for purification of bGIP and the fragmentation and identification of peptide fragments has recently 'Requests for reprints should be addressed to Maura Maletti.
75
M A L E T T I ET AL.
76
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FIG. 1. Competitive inhibition of ~I-GIP 1-42 binding by unlabeled bG1P 1-42 and its fragments 4-42, 17-42, 1-16, 1%30 and 1-3. Binding assay conditions were described in details elsewhere [8]. Each point is the mean of duplicate determinations in three separate experiments. -//
116
126
FRACTION
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146
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NUMBER
FIG. 2. Purification of ~2~I-bGIP 4-42 by HPLC. The iodination mixture resulting from the Sep-Pak C18 purification was applied to a /z-Bondapak C18 column and eluted with a gradient of CH:~CN in lC~ TFA pH 2.5.
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FIG. 3. Competitive inhibition of "-"~I-bGIP 4-42 binding by unlabeled bGIP 1-42 and its fragments 4-42, 17-42, 1-16 and 1%30. Other details as in Fig. 1.
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sion o f the rat p a n c r e a s w e r e p e r f o r m e d as p r e v i o u s l y described [4].
Preparation of Membranes M e m b r a n e s w e r e p r e p a r e d from insulin-secreting h a m s t e r p a n c r e a t i c t u m o r s . The t u m o r s [61 w e r e serially t r a n s p l a n t e d s u b c u t a n e o u s l y in Syrian h a m s t e r s [5,6]. A n i m a l s b e a r i n g
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FIG. 4. Effect of 5 ng/ml bGIP 1-42 and 17-42 on the insulin response to 6.6 mM glucose by the isolated perfused rat pancreas. Insulin response is expressed as the stimulated insulin release to basal insulin release ratio. Data are the mean of two separate experiments. The effect of 19 mM arginine (ARG) was taken as a control response.
S T R U C T U R E - F U N C T I O N R E L A T I O N S H I P OF GIP
77 RESULTS
100
Competitive Inhibition of '2"~I-GIP 1-42 by GIP Fragments
,17-42
The effect o f b G I P 1-42 and its fragments 1-3, 4-42, 1-16, 17-42 and 1%30 in competing with the binding of '2'~I-GIP 1-42 to the transplantable hamster insulinoma is shown in Fig. 1. GIP 1-42, 4-42 and 17-42 competitively inhibited the binding of the tracer. Half maximal inhibition of ~z'~I-GIPwas obtained at 6.6×10 ~ M, 2.2× 10 9 M and 10-6 M with GIP 1-42, 4-42 and 17-42 respectively. Fragments 1-3, 1-16 and 1%30 have no competitive effect at a concentration of 10-'~ M.
Purification of '2~I-GIP 4--42 To further investigate the similarity of bG1P 1-42 and 4-42 in interacting with the GIP receptor, we labeled bGIP 4-42 with ~Z~lto trace the reaction. '2'~I-labeled GIP 4-42 was separated in two main peaks by HPLC as shown in Fig. 2. A preferential binding tc plasma membrane of the transplantable hamster insulinomawas found with peak I of '25I-GIP 4-42, which was used for further experiments.
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, 11
The results are shown in Fig. 3. The pattern of the curves is similar to that obtained when'2'~I-GIP 1-42 was used as a tracer. Half-maximal inhibition of '2:'I-GIP was obtained at 5× 10-' M, 5× 10 " M and 5× 10-7 M with GIP 1-42 4--42 and 17-42 respectively. GIP 1-16 and 1%30 (10 5 M) were also ineffective on the binding of '~I-GIP 4-42.
[lIIIII
Biological Activity of GIP 17--42 and 4--42
,,,,,,,,
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10-s
GIP CONCENTRATION (M) FIG. 5. Comparison of bovine and porcine GIP by competitive inhibition of '2'~I-pGIPby unlabeled GIP 1-42 and its fragments 4-42 and 17-42. The stripped area represents the competition due to bGIP (mean+SEM). The inhibition curves of pGIP 1-42, 4-42 and 17-42 are indicated by: • and • respectively.
the tumor were killed approximately 60 days after grafting. The tumors were dissected, homogenized in 4 vol solution of 0.25 M sucrose, 0.01 M triethanolamine, 5 mM EDTA, pH 7.5 with a Dounce homogenizer, and centrifuged at 2,000 × g for 20 min. The supernatant was centrifuged at 20,000 × g for 20 rain; the resulting pellet was suspended in 6 mM HEPES, pH 7.5, and centrifuged at 20,000 × g for 20 min. Membrane pellets were stored at -80°C until use. Protein levels were determined by the method of Bradford [1].
Binding Assay The binding assay technique used here was essentially the same as that reported previously [8]. Briefly, membranes (0.5 mg protein/ml) were incubated with a fixed quantity of '251-GIP (about 1× 10-" M) and increasing amounts of natural GIP. The buffer was 5 mM MnCI2 in 60 mM HEPES, pH 7.5, containing 3% (w/v) BSA and 1 mg/ml bacitracin. After a 60-min incubation at 10°C, bound '25I-GIP was isolated by centrifugation and the radioactivity of the washed pellet was determined with a gamma counter. In all experiments, data are reported as specific binding by subtracting the nonspecific binding from the total, i.e., the amount of labeled peptide that was not displaced by an excess of natural GIP (10 '~ M).
As the results do not demonstrate if GIP 17-42, bound to the receptor sites, acts as an agonist or an antagonist of GIP, we have studied its effect in stimulating insulin release from the isolated perfused rat pancreas, bGIP 1-42 and 17-42 were infused for 15 min to achieve a perfusate concentration of 5 ng/ml in the presence of 6.6 mM glucose, according to the procedure previously described to test GIP fragments [13]. Both peptides produced an increase in insulin release (Fig. 4). The effect of the GIP 17-42 fragment corresponded to 32% of GIP 1-42 activity. This indicates that the 17-42 fragment is not an antagonist of GIP in this system. Infusion of bGIP 4--42 in six rat pancreas, under the same conditions, resulted in a small stimulation of insulin release as compared to bGIP 1-42 in only half of them. The biological activity of bGIP 1-16 and 1%30 was not tested, because of the complete loss of binding property found with these fragments (Fig. 1) and the loss of insulinotropic activity previously observed with pGIP 1-14 [13].
Comparison of Bovine and Porcine GIP by Binding Experiments Bovine and porcine GIP were compared using '2~I-pGIP as a tracer. As shown in Fig. 5, no difference was observed between the porcine and bovine GIP 1-42, 4-42, and 17-42, suggesting that the residue which is different (Ile-37 instead of Lys-37) does not induce any modification of the binding of GIP to the receptor. DISCUSSION
The comparison of the biological activity of GIP with its fragments has not yet been carried out because of difficulties in obtaining the fragments in sufficient amounts for a complete dose effect.
78
MALETTI E T A L .
In the results presented here two different systems have been used to characterize the GIP binding sites and the biological activity of GIP. Our data indicate that GIP 17-42 retains the property to bind the GIP receptor and, at least partly, to stimulate insulin release. It should be noted that high doses of GIP 17-42 are needed for competing with v-"~IGIP, whereas an insulinotropic activity of the fragment is observed at doses as low as 5 ng/ml. This suggests that even a low affinity binding of the ligand to the receptor could ensure a biological effect. Previous study on the effect of pGIP fragments on insulin release has shown that the C-terminal GIP 15-43 at a dose level of 10 ng/ml is about 40% as potent as intact GIP at 5 ng/ml, in the presence of 8.9 mM glucose [13]. The present results demonstrate that the activity of GIP 4-42, when observed, is slight, corresponding to approximately 10% of that of GIP 1-42. The absence of any insulinreleasing effect was noticed previously for pGIP 3-42, when infused in rat at a concentration of 5 ng/ml, in the presence of 8.8 mM glucose [2,10]. Our data suggest that the binding properties of GIP 4-42 are not directly related to its ability to stimulate insulin release. Indeed GIP 4-42, which competes as well as GIP 1-42 with '2~I-GIP binding, produces a small increase in insulin release, if any. In this regard, fragment 4-42 behaves like a partial antagonist of GIP. By contrast, it is of interest that suppression of the C-terminal part of pGIP as in GIP 1-38 did not result in any loss of the insulinotropic activity [12]. Shortening GIP to a
1%30 fragment suppresses the binding property of the molecule. Similarly, no effect was found with GIP 1-16 fragment. This result is supported by the complete loss of insulinotropic activity previously observed with pGIP 1-14 [131. Therefore the shortest GIP fragment showing both binding and insulinotropic activity is the 17-42 fragment. The present results, together with the effect of GIP 1-38 in stimulating insulin release [12], stress that the structural requirements for the binding and biological activity of the molecule are contained within the sequence 17-38. Interesting, however, is the observation that the differences between the porcine, bovine and human [11] GIP molecules are located in this 17-38 sequence, in positions 37 (bovine) or 18 and 34 (human). Our present findings also suggest that the N-terminal portion of GIP can be removed without impairing the activity of the molecule. Further studies, using G | P modified in the C-terminal part, are required to get further insight into the structurefunction of the interaction of GIP with its receptor. ACKNOWLEDGEMENTS This work was supported by grants from the Swedish Medical Research Council (project No. 01010) and the Nordisk lnsulinfond. We wish to thank Drs. J. Grenier, K. Nahoul and M. Roger for critical review of the manuscript, D. Bailbe, G. Cattoire and R. Victor Rapharl for their excellent technical assistance.
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8. Maletti, M., B. Portha, M. Carlquist, M. Kergoat, M. Laburthe, J. C. Marie and G. Rosselin. Evidence for and characterization of specific high affinity binding sites for the gastric inhibitory polypeptide in pancreatic r-cells. Endoerinalo~,y 115: 13241331, 1984. 9. Marie, J. C., C. Boissard and G. Rosselin. Purification of monoiodinated vasointestinal peptide (MIZ~I-VIP)by high pressure liquid chromatography (HPLC). Peptides 5: 17%182, 1984. 10. Moody, A. J., K. Damm J0rgensen and L. Thim. Structurefunction relationships in porcine GIPDiabetolozia 21: 306, 1981 (abstr). 11. Moody, A. J., L. Thim and 1. Valverde. The isolation and sequencing of human gastric inhibitory peptide (GIP). FEBS Lett 172: 142-148, 1984. 12. Moroder, L., A. Hallet, P. Thamm, L. Wilschowitz, J. C. Brown and E. WOnsch. Studies on gastric inhibitory polypeptide: Synthesis of the octatriacontapeptide GIP 1-38 with full insulinotropic activity. Seand J Gastroenterol 13: Suppl 49, 129, 1978. 13. Pederson, R. A. and J. C. Brown. The insulinotropic action of gastric inhibitory polypeptide in the perfused isolated rat pancreas. Endocrinology 99: 780-785, 1976.