Intracellular localization and molecular heterogeneity of the sulphonylurea receptor in insulin-secreting cells

Diabetologia, Mar 1995

Sulphonylureas stimulate insulin secretion by binding to a receptor in the pancreatic beta-cell plasma membrane resulting in inhibition of ATP-sensitive K+ channels, membrane depolarization and thus influx of Ca2+ through voltage-dependent Ca2+ channels. Sulphonylureas can also induce hormone release at fixed membrane potentials without Ca2+ entry suggesting that these drugs may have other modes of action. We have determined whether different forms of sulphonylurea-binding proteins are present in insulin-secreting cells and their subcellular localization by density gradient centrifugation. Binding studies using [3H]-glibenclamide showed that islet and insulinoma membranes contained a single high affinity sulphonylurea binding site (Kd = 1 nmol/l). Photo-crosslinking of the drug to the membranes resulted in labelling of two proteins with apparent molecular weights of 170 and 140 kDa. The same analyses of insulinoma subcellular fractions showed that the majority (>90%) of binding proteins were localized to intracellular membranes with only minor levels (<10%) on plasma membranes. The 170 kDa sulphonylurea binding protein was present in both plasma and granule membrane fractions whereas the 140 kDa form was not present in the plasma membrane fraction. The differences in the molecular forms and subcellular distribution of the receptor are consistent with sulphonylureas having multiple sites of action in the pancreatic beta cell.

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Intracellular localization and molecular heterogeneity of the sulphonylurea receptor in insulin-secreting cells

Diabetologia Intracellular localization and molecular heterogeneity of the sulphonylurea receptor in insulin-secreting cells S. E. Ozanne 0 P. C. Guest 0 J. C. Hutton 0 C. N. Hales 0 0 Department of ClinicalBiochemistry,University of Cambridge, Addenbrooke's Hospital , Cambridge , UK Summary Sulphonylureas stimulate insulin secretion by binding to a receptor in the pancreatic beta-cell plasma m e m b r a n e resulting in inhibition of ATP-sensitive K + channels, membrane depolarization and thus influx of Ca2§ through voltage-dependent Ca2+ channels. Sulphonylureas can also induce hormone release at fixed m e m b r a n e potentials without Ca2+ entry suggesting that these drugs may have other modes of action. We have determined whether different forms of sulphonylurea-binding proteins are present in insulin-secreting cells and their subcellular localization by density gradient centrifugation. Binding studies using [3H]-glibenclamide showed that islet and insulinoma membranes contained a single high affinity sulphonylurea binding site (Kd= 1 nmol/1). Photo-crosslinking of the drug to the membranes resulted in labelling of two proteins with apparent molecular weights of 170 and 140 kDa. The same analyses of insulinoma subcellular fractions showed that the majority ( > 90 %) of binding proteins were localized to intracellular membranes with only minor levels ( < 10 %) on plasma membranes. The 170 k D a sulphonylurea binding protein was present in both plasma and granule m e m b r a n e fractions whereas the 140 kDa form was not present in the plasma membrane fraction. The differences in the molecular forms and subcelltflar distribution of the receptor are consistent with sulphonylureas having multiple sites of action in the pancreatic beta cell. [Diabetologia (1995) 38: 277-282] Non-insulin-dependent diabetes mellitus; insulin; sulphonylurea receptors; islets; glibenclamide; secretory granule - 9 Springer-Verlag1995 Sulphonylureas have been used to treat non-insulindependent diabetic patients for over 30 years. These drugs stimulate insulin secretion by inhibiting ATPsensitive potassium (KATP)channels which leads to depolarization of the plasma membrane, activation of voltage-dependent Ca2+ channels and an increase in cytoplasmic Ca2+ levels [ 1 ]. High-affinity sulphonylurea binding sites have been reported in a number of insulin-secreting cells including H I T cells and murine islets [ 2-4 ]. Photoaffinity labelling studies using [3H]-glibenclamide [ 5 ] and [125I]-glibenclamide [ 6, 7 ] have shown that the binding protein has an apparent molecular weight of 140 kDa in H I T cells, as determined by SDS-PAGE under reducing conditions. Recent radiation inactivation studies have shown that the native sulphonylurea receptor in MIN6 beta-cell membranes is a multimer with a molecular mass of 250 kDa [8]. The mechanism by which sulphonylurea binding elicits KATp-channel inhibition has not been elucidated, although patch clamp studies have shown that the process is Mg2+dependent [ 9 ]. Studies combining patch clamp, drug binding and secretion analyses of the CRI-G1 and C R I - D l l cell lines, which differ in their sensitivity to sulphonylureas, have shown that the receptor and channel moieties are closely-associated in the plasma m e m b r a n e although they appear to reside on separate polypeptide chains [ 10 ]. Apart from the inhibitory effect of sulphonylureas on plasma membrane KATP channels, sulphonylureas may stimulate insulin secretion by other mechanisms. Studies using electrically-permeabilized RINm5f cells have shown that tolbutamide can stimulate insulin secretion by a mechanism which is not dependent on changes in m e m b r a n e potential [ 11 ]. Similarly, A m m a l a et al. [ 12 ] have shown that tolbutamide enhanced the rate of exocytosis from depolarized mouse beta cells without any change in the existing Ca2+ current. Since ultrastructural studies show that [3H]-glibenclamide associates with secretory granules in rat islets of Langerhans [ 13 ], it is proposed that the drug may interact with the secretory machinery. However, the nature of such intracellular sulphonylurea binding sites and their relationship to the receptor involved in KATP channel regulation have not been characterized. The aim of the present study was to investigate the molecular complexity of sulphonylurea binding proteins in insulin-secreting cells by glibenclamide binding studies and photoaffinity-tabelling analyses. The subcellular distribution of different molecular forms was determined by density gradient centrifugation analyses of transplantable insulinoma tissue. Materials and methods Materials. Analytical grade biochemicals were obtained from Sigma Chemical Co. or BDH Chemicals (both of Poole, Dorset, UK), unless stated otherwise. [3H]-glibenclamide (50.7 Ci/ retool) was obtained from NEN-Dupont (Stevenage, Herts., UK). Tissues. Insulinomas were propagated in New England Deaconess Hospital (NEDH) rats as described previously [ 14 ]. Tumours were removed and tissue was scraped from the fibrous capsules into Hanks saline [(in retool/l) 137 NaC1, 5.4 KC1, 1.67 MgSO 4, 4 CaC12, 0.34 NazHPO4 and 4.2 NaHCO 3 (pH 7.4)]. All subsequent procedures were performed at 4 ~ Cells were isolated from insulinoma tissue by Percoll density gradient centrifugation as described previously [ 15 ] and used immediately. The CRI-G1 and C R I - D l l insulin-secreting cell lines, derived initially from transplantable rat insulinoma cells, were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10 % fetal calf serum (both from Flow Laboratories, Irvine, Scotland, UK) and passaged at 4-7 day intervals as described by Carrington et al. [ 16 ]. Islets of Langerhans were isolated from the pancreata of 8-12-week-old N E D H rats by collagenase digestion followed by purification on Histopaque density gradients as described by Guest et al. [ 17 ]. Islets were cultured overnight in DMEM containing 11 mmol/1 glucose and 10 % fetal calf serum. Subcellular fractionation of insulinomas. Insulinoma tissue was homogenized in isotonic media and fractionated by density gradient centrifugation on Nycodenz step gradients as described by Hutton et al. [ 18 ]. This yielded four membrane fractions designated as A, B, C and D in order of increasing density. Fractions were stored in the presence of 100 ~tmol/1phenylmethylsulphonyl fluoride (PMSF), 20 ~mol/1 trans-EpoxyS. E. Ozanne et al.: Sulphonylurea receptors in pancreatic beta cells succinyl-L-leucylamido(4-guanidino)-butane (E-64) and 10 ~mol/1 pepstatin A under liquid nitrogen prior to analysis. Membrane preparation. Islets, CRI-G1 and C R I - D l l cells were lysed and crude membrane fractions prepared by ultracentrifugation as described previously [ 10 ]. Samples were resuspended in 50mmol/1 Tris/HC1 (pH7.4) containing 100 retool/1 PMSF, 10 mmol/1 Pepstatin A and 20 retool/1 E64 and stored under liquid nitrogen prior to use. Salt washing of insulinoma membranes. Insulinoma membrane fractions were pelleted by centrifugation for 30rain at 100,000 x g (Sorvall T-1270; Du Pont (U. K) Ltd., Stevenage, Merts, UK), then resuspended in 2ml of 10mmol/1 NHaHCO3 and sonicated for 10 s at 3 ~m (MSE Sonifier, Crawley, Sussex, UK). Centrifugation was repeated and the pellets were resuspended in 2 ml of 10 mmol/1 NH4HCO 3 containing 1 mol/1 NaC1 and 1 mmol/1 EDTA, sonicated and centrifuged as above. The pellet was resuspended in 2 ml of 10 mmol/1 NH4HCO 3 and the sonication and centrifugation steps were repeated. The membranes were stored under liquid nitrogen in 1 ml of 50 mmol/1 Tris/HC1 pH 7.4 containing the protease inhibitors described above. Control (non-salt washed) membranes were treated in an identical manner except all procedures were performed in 50 mmol/1 Tris/HC1 (pH 7.4). [~H]-glibenclamide binding studies. Membranes prepared from islets, insulinoma, CRI-G1 and C R I - D l l cells (25 ~tgprotein) were incubated for 2 h at room temperature in 0.5 ml assay buffer (50 mmol/1 Tris/HC1 pH 7.4) containing the indicated concentrations of [3H]-glibenclamide (50.9 Ci/mmol, Du Pont [UK] Ltd). Incubations were terminated by addition of 2 ml of ice-cold buffer and bound ligand was collected by rapid vacuum filtration onto 2.5-cm diameter GF/B filter disks (Whatman International Ltd, Maidstone, Kent, UK). Filters were washed with 4 x 2 ml of ice-cold buffer and the bound radioactivity determined by liquid scintillation counting using OptiPhase HiSafe II (LKB Scintillation Products, Loughborough, Leicestershire, UK) and a Packard liqnid-scintillation spectrometer (Canberra Packard, Slaugh, Berkshire, UK). Assay protocols. Protein was determined by a dye binding method using bovine serum albumin as standard [ 19 ]. Insulin was measured by radioimmunoassay as described previously [ 20 ]. Spectrophotometric assays were performed for cytochrome oxidase [ 21 ], NADPH-cytochrome c reductase [ 22 ] and alkaline phosphatase [ 23 ]. /3-N-acetylglucosaminidase was determined by a fluorometric method [ 24 ]. Photoaffinity labelling. Membrane fractions from each cell type (100 ~tgprotein) were incubated for 2 h at room temperature in 60 ~tl of sodium phosphate buffer (pH 7.5) containing 10 nmol/1 [3H]-glibenelamide. The samples were irradiated for 15 min at 312 nm in a Stratalinker (Stratagene, La Jolla, Calif., USA) and then combined with an equal volume of 2 x concentrated sample loading buffer (0.25 tool/1 Tris/HC1 pH 9.2 % SDS, 12 % sucrose, 20 mmol/1 EDTA, 130 mmol/1 dithiothreitol). Samples were subjected to SDS-polyacrylamide gel electrophoresis on slab gels (15 x 15 0.15 cm) polymerized from 10 % (weight/volume) acrylamide and 0.13 % NN'-methylenebisacrylamide using the discontinuous buffer system of Laemmli [ 25 ]. Gels were impregnated for fluorography with 20 % (weight/volume) 2, 5-diphenyloxazole in acetic acid, vacuum dried and exposed to Kodak Scientific Imaging Film (Eastman Kodak Company, New York, N.Y., USA) at 70 ~C. Results Binding studies on total membranes. High affinity glibenclamide binding sites were detected in total m e m b r a n e s prepared from transplantable rat insulinoma cells (Fig. 1). The affinity of the insulinoma receptors (1.17 + 0.18 nmol/1) was similar to those expressed in the CRI-G1 cell line (Kd=1.31+ 0.06 nmol/1), although the Bmax was approximately three-fold higher (4.3 _+0.2 vs 1.4 + 0.1 pmol/mg). Washing of the insulinoma m e m b r a n e s with NaC1and EDTA-containing solutions had no effect on the affinity of the receptor for glibenclamide (Kd of 1.23 + 0.3 nmol/1) and only slightly reduced the number of receptors detected (Bmax of 3.8 _+0.1 pmol/mg initial protein). However, the specific activity of receptor binding increased from 4.3 + 0.2 to 16.5 + 0.5 pmol/mg following m e m b r a n e washing. Subcellular fractionation o f insulinoma membranes. Nycodenz density gradient centrifugation of insulin o m a tissue produced four m e m b r a n e fractions designated A, B, C and D, in order of increasing density. Analysis of selected m a r k e r proteins showed that f r a c t i o n A was enriched in plasma membranes, fraction B was a mixture of plasma m e m b r a n e s and secretory granules, fraction C was enriched in secretory granules and fraction D was a mixture of granules, mitochondria, lysosomes and endoplasmic reticulum (ER) (Table 1). Glibenclamide binding studies showed that the sulphonylurea receptor was distributed across the gradient with slightly higher specific activities detected in fractions B and C (Table 1). Thus, from total recoveries of each fraction it was estimated that fractions A, B, C and D contained 1 % , 3 %, 30 % and 66 % of the total recovered cellular binding sites, respectively. This was similar to the distribution of insulin secretory granules as fractions A, B, C and D contained 0.5 %, 2 %, 35 % and 62.5 % of the total recovered immunoreactive insulin, respectively, whereas E R , mitochondria (1) secretory granules (insulin), (2) lysosomes (/%N-acetylglucosaminidase), (3) mitochondria (cytochromec oxidase), (4) Endoplasmic reticulum (NADPH-cytochromec reductase) and (5) plasma membranes (alkaline phosphatase) sooo] Islets Insulinoma CRI-G1 CRI-D11 Fig.2. Comparison of insulin content and sulphonylurea binding in insulin-secreting cells. The indicated membranes were incubated in 0.2 nmol/1 [3H]-glibenclamide for 2 h and then bound radioactivity was collected by vacuum filtration onto GF/B disks and determined by liquid scintillation counting as described in the methods section. Non-specific binding (NSB) was determined in the presence of 1 ~mol/1glibenclamide. Results are expressed as fmol bound/mg protein after subtraction of NSB and are the mean + SEM of three experiments. Insulin was determined by radioimmunoassay and results are the mean + SEM of three experiments each performed in triplicate Sulphonylurea receptors; Insulin and lysosomes s h o w e d a different distribution (Table 1). Insulin and sulphonylurea receptor levels in different beta-cell sources. Insulin r a d i o i m m u n o a s s a y and glibenclamide binding studies s h o w e d that there was a strong correlation (R = 0.92) b e t w e e n cellular insulin content and n u m b e r of sulphonylurea receptors present in the corresponding total cellular m e m branes with the highest levels of each f o u n d in pancreatic islets and the lowest f o u n d in C R I - D l l cells (Fig.Z). Photoaffinity labelling studies. Ultraviolet-crosslinking of [3H]-glibenclamide to m e m b r a n e s prep a r e d f r o m pancreatic islets, insulinoma and C R I - G 1 cells labelled two proteins of a p p a r e n t molecular weights 140 k D a and 170 k D a (Fig.3). There was a difference in the relative levels of these two proteins in the different cell types. In C R I - G 1 cells the 140 k D a species was the p r e d o m i n a n t component, whereas the 170 k D a f o r m was m o r e a b u n d a n t in islets and insulinoma tissue. The two forms of the rec e p t o r w e r e also distributed differently in different insulinoma subcellular m e m b r a n e fractions (Fig.4). The plasma m e m b r a n e - and secretory granule- enriched fractions A, B and C contained only the 170 k D a f o r m of the r e c e p t o r whereas fraction D, which contained lysosomes, mitochondria and E R in addition to secretory granules, had b o t h the 170 k D a and 140 k D a forms of the receptor. N e i t h e r of the 500 m Fig. 3. Photoaffinity labelling of sulphonylurea receptors present in islets, insulinoma and CRI-G1 membranes, Membranes (50 ~tg) were incubated with 10 nmol/l [3H] glibenclamide and irradiated at 302 nm for 15 min as described in the methods section. Labelled proteins in islets (track 1), insulinoma (track 2) and CRI-G1 (track 3) membranes were detected by SDS-PAGE and fluorography proteins were labelled in the presence of i ~tmol/1 unlabelled glibenclamide (Fig. 4). Discussion Sulphonylurea receptors have b e e n identified in a variety of tissues including pancreatic b e t a cells, ventricular m y o c y t e s and cerebral cortex [ 1 ]. The molecular structure of the r e c e p t o r is p o o r l y understood, although it has b e e n suggested that it is glycated b a s e d on its ability to bind w h e a t g e r m agglutinin and its a p p a r e n t increase in mobility on S D S - P A G E gels following t r e a t m e n t with e n d o g l y c o s i d a s e F [ 26 ]. The present study s h o w e d that tumours propagated in N E D H rats express sulphonylurea receptors with a single t y p e of high affinity binding site. These receptors remain associated with the m e m b r a n e following stringent washing p r o c e d u r e s suggesting that t h e y might b e either integral m e m b r a n e proteins or covalently attached to the m e m b r a n e . This observation is consistent with recent structural studies which p r o p o s e d that the r e c e p t o r contains multiple transm e m b r a n e spanning domains [ 27 ]. Sulphonylurea binding studies of insulinoma subcellular fractions showed that there was a high specific activity of receptors on secretory granule membranes. This is consistent with the observation that the levels of sulphonylurea receptors and insulin were correlated in a n u m b e r of different sources of beta cells which differ markedly in their extent of granulation. The finding that most of these binding sites were intracellular is consistent with previous studies which have shown that the receptors are not sensitive to trypsinolysis of intact cells [ 28 ]. These findings support the hypothesis that sulphonylurea receptors might regulate insulin secretion by m o r e than one mechanism and raises the possibility that this could occur through direct interaction of the receptor with granule m e m b r a n e proteins such as those involved in granule m o v e m e n t or m e m b r a n e fusion. Little is known about the structural organization of the sulphonylurea receptor in membranes. Numerous studies employing photoaffinity labelling have shown that the high affinity sulphonylurea binding site is present on a polypeptide chain with an apparent molecular weight of 140 k D a to 150 k D a [ 5-7, 29 ]. The present photoaffinity labelling approach demonstrated that there are actually two species of sulphonylurea receptor with molecular weights of approximately 140 and 170 k D a in crude m e m b r a n e preparations of rat islet. CRI-G1 and insulinoma cells. The subcellular distribution of these components in the insulinoma tissue was consistent with that of sulphonylurea binding sites, with a predominant localization to intracellular membranes. A small proportion of the 170 k D a form of the receptor was found in association with subcellular fractions enriched in plasma m e m b r a n e and the 140 k D a form was present only in the subcellular fraction associated with dense organelles. The relationship between the two receptors is not clear. It is possible that they are two forms of the same protein which arise from either differential m R N A splicing or post-translational modifications. It is unlikely that the 140 k D a species arises from non-specific proteolysis of the 170 k D a protein because protease inhibitors were present throughout m e m b r a n e preparation and the relative proportion of the proteins was similar in separate experiments. Electrophysiological studies have shown that application of sulphonylureas to excised plasma m e m b r a n e patches results in inhibition of KATp channel activity [ 30 ]. The presence of the 170 k D a form of the receptor in a subcellular fraction enriched in plasma m e m branes suggests that it might be the form responsible for regulation of KaTe channels. It is less clear why most of the 170 k D a form and all of the 140 k D a receptor are localized to fractions containing a mixture of granules, mitochondria, E R and lysosomes. O n e possibility is that one or both of the receptors directly mediate exocytotic release of insulin. A n o t h e r explanation is that these receptors could be associated with granule m e m b r a n e ion channels which are involved in the regulation of the intragranular environment. Sulphonylurea-sensitive K + identified previously on mitochondrial m e m b r a n e s [ 31 ] and zymogen granules [ 32 ]. It has been suggested that these channels m a y play an important role in the fusion of zymogen granule m e m b r a n e s with the plasma membrane. It is possible that sulphonylureas also regulate the fusion of insulin secretory granule m e m b r a n e s with the plasma membrane. The presence of a large intracellular pool of sulphonylurea receptors m a y also provide a versatile mechanism by which interaction of sulphonylurea receptors with plasma m e m brane KATP channels could be regulated. For example, stimulation of the cell might result in exocytotic deposition of the granule m e m b r a n e proteins in the vicinity of KATP channels, and removal of the stimulus might lead to their endocytotic retrieval for intracellular storage. Further studies are warranted to test these possibilities and to determine whether the two forms of the receptor have different activities. Acknowledgements. This work was supported by grants from the British Diabetic Association, the Medical Research Council of Great Britain and the Wellcome Trust. 1. Ashcroft SJH , Ashcroft FM ( 1992 ) The sulphonylurea receptor . Biochim Biophys Acta 1175 : 45 - 59 2. Geisen K , Okomonopoulos HR , Punter J , Weyer R , Summ H-D ( 1985 ) Inhibition of 3H-glibenclamide binding to sulphonylurea receptors by oral antidiabetics . Drug Res 35 : 707 - 712 3. Gaines KL , Hamilton S , Boyd AE ( 1988 ) Characterisation of the sulphonylurea receptor on/3-cell membranes . J Biol Chem 263 : 2589 - 2592 4. 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Dr. S. E. Ozanne, P. C. Guest, J. C. Hutton, C. N. Hales. Intracellular localization and molecular heterogeneity of the sulphonylurea receptor in insulin-secreting cells, Diabetologia, 1995, 277-282, DOI: 10.1007/BF00400631