The GIP receptor on pancreatic beta cell tumor: molecular identification by covalent cross-linking

125I-GIP binds reversibly to a high affinity binding site in crude plasma membranes prepared from a hamster pancreatic beta cell tumor. The treatment of labeled membranes with the cross-linker dithiobis (succinimidylpropionate) prevents, to a greater extent, the rapid dissociation of 125I-GIP-membrane complexes which is observed when 10(-6) M native GIP is added. Polyacrylamide gel electrophoresis of membrane proteins reveals a major 125I-GIP-protein complex of Mr 64,000. This labeling decreases when increasing concentrations (10(-9) -10(-6)M) of native GIP are added but is not altered by other peptide hormones (tested at 10(-6)M) including glucagon, VIP and insulin. The Mr 64,000 complex is not observed in tissues which have no specific binding sites for GIP such as intestinal epithelium. Assuming one molecule of 125I-GIP is bound per molecule of protein, one protein with Mr 59,000 is identified as the specific GIP binding site.     

Somatostatin receptors on rat cerebrocortical membranes. Structural characterization of somatostatin-14 and somatostatin-28 receptors and comparison with pancreatic type receptors

Somatostatin binding and cross-linking to its receptors on rat cerebrocortical membranes were characterized with [125I-Tyr1]somatostatin-14 and [125I-Leu8, D-Trp22, Tyr25]somatostatin-28. When [125I-Tyr1]somatostatin-14 was cross-linked to its receptors with the photoreactive cross-linker, N-(5-azido-2-nitrobenzoyloxy)succinimide, the hormone was specifically associated with a Mr = 72,000 protein band in the presence or absence of reducing agents. Affinity labeling of the Mr = 72,000 protein band was decreased with increasing concentrations of unlabeled somatostatin-14 and nonhydrolyzable guanine nucleotide analog, guanyl-5'-yl imidodiphosphate (Gpp(NH)p). Pretreatment of cerebrocortical membranes with islet-activating protein resulted in a decrease in subsequent labeled somatostatin-14 binding and affinity-labeling of the protein and abolished an inhibitory effect of somatostatin-14 on vasoactive intestinal peptide-stimulated increase in adenylate cyclase activity. When the affinity-labeled protein was solubilized with Zwittergent 3-12 and adsorbed to wheat germ agglutinin-agarose, it was eluted by N-acetylglucosamine. [125I-Leu8, D-Trp22, Tyr25]somatostatin-28 cross-linking to cerebrocortical and pancreatic membranes with the same photoreactive agent revealed specifically labeled protein bands of a Mr = 74,000 in cerebrocortical membranes and a Mr = 94,000 in pancreatic membranes, respectively. These results suggest that: 1) somatostatin receptor on cerebrocortical membranes is a monomeric glycoprotein with a Mr = 70,000 binding subunit, coupled to guanine nucleotide regulatory protein, and 2) the Mr = 70,000 protein may be a common receptor for somatostatin-28 and somatostatin-14 and is distinct from a common pancreatic type receptor.     

Identification of the glucagon receptor by covalent labeling with a radiolabeled photoreactive glucagon analogue

The photoreactive 125I-labeled glucagon-NAPS [125I-labeled 2-[2-nitro-4-azidophenyl)sulfenyl]-Trp25-glucagon] was used to label the glucagon receptor sites in rat liver plasma membranes. The proteins labeled were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with or without reduction with dithiothreitol. The photoaffinity peptide specifically labeled a number of bands with apparent molecular weights greater than 200000 and probably at least two protein bands in the molecular weight range 52000-70000. The relative amounts of radioactivity associated with these bands and their relative mobilities differed in samples from reduced and unreduced membranes. Their relative mobilities also differed with percent acrylamide cross-linking, suggesting a glycoprotein nature and the presence of intramolecular disulfide bonds. A nonspecifically labeled band with an apparent molecular weight of 27000-28000 also displayed a similar behavior. Photolabeling in the presence of 0.1 mM guanosine 5'-triphosphate (GTP) decreased the amount of radiolabeling of these bands, suggesting their involvement in the glucagon stimulation of adenylate cyclase. The photolabeled receptor in the membranes, solubilized with Lubrol-PX and fractionated on an Ultrogel AcA22 column, eluted with an apparent molecular weight of 200000-250000. Addition of GTP to the solubilized glucagon receptor of nonirradiated membranes caused complete dissociation of the complex. Gel electrophoresis of the partially purified radiolabeled receptor identified the same protein components observed in photolabeled membranes. These results indicate that the glucagon receptor is an oligomer probably composed of at least two different subunits that are linked together or greatly stabilized by disulfide bonds. They also show that 125I-labeled glucagon-NAPS can be used effectively to covalently label the putative glucagon receptor and thus aid in its further characterization.     

The somatostatin receptor on isolated pancreatic acinar cell plasma membranes. Identification of subunit structure and direct regulation by cholecystokinin

Somatostatin binding to its receptors on rat pancreatic acinar membranes was characterized with [125I-Tyr1]somatostatin. Binding at 24 degrees C was rapid reaching a maximum after 60 min and was reversible upon the addition of 1 microM unlabeled ligand. Scatchard analysis revealed a single class of binding sites, with a Kd of 0.32 +/- 0.03 nM and a binding capacity of 600 +/- 54 fmol/mg of protein. Specificity for the somatostatin was demonstrated with the inhibition of labeled hormone binding by somatostatin analogs in proportion to their biological activities. When [125I-Tyr1]somatostatin was cross-linked to its receptors with the photoreactive cross-linker n-hydroxysuccinimidyl-4-azidobenzoate, the hormone was associated with Mr = 90,000 protein. Similar mobilities of the radioactive band were observed in the presence and absence of dithiothreitol. In contrast to other unrelated peptides, cholecystokinin (CCK) and its analogs directly reduced [125I-Tyr1] somatostatin binding to isolated membranes. The effect of CCK was one-half-maximal at 3 nM and maximal at 100 nM. In the presence of 3 nM CCK8, the binding capacity for somatostatin was decreased to 237 +/- 39 fmol/mg of protein without a significant change in affinity. Dibutyryl cyclic GMP, a CCK receptor antagonist, blocked this action of CCK8 indicating that the CCK receptor mediated the decrease in [125-Tyr1]somatostatin binding. In contrast cerebral cortex membranes, which also possess a somatostatin receptor, were not regulated by CCK. These results indicate, therefore, that 1) purified pancreatic acinar plasma membranes contain specific receptors for somatostatin, 2) the receptor has an apparent Mr of about 90,000, and 3) the binding of somatostatin to its receptor on pancreatic plasma membranes is regulated by CCK analogs acting via the CCK receptor.     

Characterization of the bombesin receptor on mouse pancreatic acini by chemical cross-linking

Bombesin (BN), gastrin-releasing peptide (GRP) and GRP(18–27) (neuromedin C) were equipotent and 30-fold more potent than neuromedin B (NMB) in inhibiting binding of ¹²⁵I-GRP to and in stimulating amylase release from mouse pancreatic acini. In the present study we used ¹²⁵I-GRP and chemical cross-linking techniques to characterized the mouse pancreatic BN receptor. After binding of ¹²⁵I-GRP to membranes, and incubation with various chemical cross-linking agents, cross-linked radioactivity was analyzed by SDS-PAG electrophoresis and autoradiography. With each of 4 different chemical cross-linking agents, there was a single broad polypeptide band of Mr 80,000. Cross-linking did not occur in the absence of the cross-linking agent. Cross-linking was inhibited only by peptides that interact with the BN receptor such as GRP, NMB, GRP(18–27) or BN. Dose-inhibition curves for the ability of BN or NMB to inhibit binding of ¹²⁵I-GRP to membranes or cross-linking to the 80,000 polypeptide demonstrated for both that BN was 15-fold more potent than NMB. The apparent molecular weight of the cross-linked polypeptide was unchanged by adding dithiothreitol. N-Glycanase treatment reduced the molecular weight of the cross-linked peptide to 40,000. The present results indicate that the BN receptor on mouse pancreatic acinar cell membranes resembles that recently described on various tumor cells in being a single glycoprotein with a molecular weight of 76,000. Because dithiothreitol had no effect, this glycoprotein is not a subunit of a larger disulfide-linked structure.     

Characterization of the detergent solubilized receptor for gastrin-releasing peptide

Properties of detergent solubilized gastrin-releasing peptide receptor were investigated. Swiss 3T3 membranes were covalently labeled with [125I]GRP and homobifunctional cross-linkers. A major labeled protein of 75 kDa was resolved using SDS-polyacrylamide gel electrophoresis. When the same preparation was solubilized with zwitterionic detergent and analyzed under nondenaturing conditions the protein bound radioactivity was resolved in two different peaks, a major one of apparent molecular weight 220,000 (peak 1) and a minor one of 80,000 (peak 2) both containing the 75 kDa protein. Specific ligand binding activity also eluted with peak 1. These results indicate that the active form of bombesin/GRP receptor is a large complex containing the 75 kDa ligand binding domain.     

Crosslinking of HCG to its specific receptor of porcine ovarian membranes

HCG receptors of porcine ovarian plasma membranes were loaded with 125I-hCG and covalently crosslinked with glutaraldehyde. The plasma membranes were labeled with tritium. The hCG receptor complex was solubilized in 1% SDS and subsequently chromatographed on Biogel P 300. 40-50% of 125I-radioactivity eluted near the void volume. 3H radioactivity in these fractions indicated the presence of crosslinked hormone receptor complexes. A second minor peak of 125I-radioactivity appeared at 55000 d. A third peak represented subunits of 125I-hCG. Analysis of the Biogel P 300 purified 125I-hCG receptor complex by polyacrylamide gel electrophoresis in sodium dodecyl sulfate resulted in two radioactivity peaks. The main peak corresponded to 90 000-100 000 d, the second peak was calculated to approximately 140 000 d. As a result the majority of the hormone receptor complex has a molecular weight of 90 000-100 000 d. Existence of subunits is discussed.     

Solubilization and characterization of guinea-pig pancreatic somatostatin receptors

The solubilization of somatostatin receptors from guinea-pig pancreas by different non-denaturing detergents was investigated after stabilization of the receptors by prior binding of 125I-[Tyr11]somatostatin or its analogue 125I-[Leu8,DTrp22,Tyr25]somatostatin 28, to pancreatic plasma membranes. The somatostatin-receptor complexes were solubilized in a high yield by Zwittergent 3-14 (3-[tetradecyldimethylammonio]-1-propanesulfonate), a zwitterionic detergent. Other detergents, digitonin, Triton X-100, Chaps (3-[cholamidopropyldimethylammonio]-1-propanesulfonate) and octyl beta-D-glycopyranoside, achieved only partial solubilization. The recovery of receptor complexes was increased by glycerol. In order to characterize solubilized somatostatin-receptor complexes, membranes receptors were covalently labelled using N-5-azido-2-nitrobenzoyloxysuccinimide as cross-linking reagent before solubilization. Gel filtration chromatography analysis resulted in the identification of a major protein component of apparent Mr = 93,000 which interacted with the two radioligands. In addition, a similar component of Mr = 88,000 was characterized after analysis by SDS-PAGE of membrane receptors covalently cross-linked with 125I-[Leu8,DTrp22,Tyr25]somatostatin 28 by different heterobifunctional reagents: N-5-azido-2-nitrobenzoyloxysuccinimide, N-hydroxysuccinimidyl 4-azidobenzoate, N-succinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate. Optimal cross-linking results were obtained with N-5-azido-2-nitrobenzoyloxysuccinimide. The solubilized somatostatin-receptor complex was adsorbed to wheat-germ agglutinin-agarose column and eluted by specific sugars. We concluded that the guinea-pig pancreatic somatostatin receptor in the membrane and in the non-denaturing detergent solution behaves as a protein monomer of apparent Mr approximately 85,000-90,000. The somatostatin receptor is a glycoprotein which contains complex-type carbohydrate chains.     

The CCK receptor on pancreatic plasma membranes: binding characteristics and covalent cross-linking

The cholecystokinin (CCK) receptor in purified plasma membranes prepared from mouse pancreatic acini had a binding affinity of 1.8 nM, an acid pH optimum between 6.0 and 6.5, and an analog specificity of CCK8 greater than CCK33 greater than desulphated CCK8 greater than CCK4. Binding of CCK to its receptor was abolished by pretreatment of plasma membranes with trypsin. When [125I]CCK was cross-linked to its receptors with disuccinimidyl suberate, and the preparation solubilized and subjected to gel electrophoresis and autoradiography, the hormone was associated with Mr 80 000 protein in both the presence and absence of the reducing agent dithiothreitol.     

Purification and characterization of the human platelet alpha 2-adrenergic receptor

Human platelet alpha 2-adrenergic receptors have been purified approximately 80,000-fold to apparent homogeneity by a five-step chromatographic procedure. The overall yield starting from the membranes is approximately 2%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioiodinated protein from purified receptor preparations shows a single major band of Mr 64,000. The specific binding activity of the alpha 2-adrenergic receptor after four chromatographic steps is 14.5 nmol/mg protein. This value is consistent with the expected theoretical specific activity (15.6 nmol/mg) for a protein with a molecular mass of 64,000 daltons if it is assumed that there is one ligand-binding site/receptor molecule. The purified protein can be covalently labeled with the alkylating alpha-adrenergic ligand, [3H]phenoxybenzamine. This labeling is specific, and it shows that the Mr 64,000 protein contains the ligand binding site of the alpha 2-adrenergic receptor. In addition, the competitive binding of ligands to the purified receptor protein shows the proper alpha 2-adrenergic specificity. The alpha 2-adrenergic receptor contains an essential sulfhydryl residue. Thus, exposure of the purified receptor to the sulfhydryl-specific reagent, phenylmercuric chloride, resulted in an 80% loss of binding activity. This loss of binding activity was prevented when exposure to phenylmercuric chloride was done in the presence of alpha 2-adrenergic ligands, and it was reversed by subsequent exposure to dithiothreitol. Partial proteolysis of purified alpha 2-adrenergic receptors was obtained with Staphylococcus aureus V-8 protease, alpha-chymotrypsin, and papain. In a comparison with purified beta 2-adrenergic receptors, no common partial proteolytic products were found.     

Functional and immunological evidence for stable association of solubilized vasoactive-intestinal-peptide receptor and stimulatory guanine-nucleotide-binding protein from rat liver

We have reported the solubilization of complexes between vasoactive intestinal peptide (VIP) and its receptor from rat liver in a GTP-sensitive form of Mr 150,000 [Couvineau, A., Amiranoff, B. & Laburthe, M. (1986) J. Biol. Chem. 261, 14482-14489]. In the present study, we demonstrate a stable association of solubilized VIP receptor and stimulatory guanine nucleotide-binding protein (Gs protein), taking advantage of the ability of the glycoproteic VIP receptor (Mr 48,000), and the inability of the Gs protein, to adsorb to wheat germ agglutinin (WGA). 125I-VIP-receptor complexes solubilized in Triton X-100 were adsorbed on WGA-Sepharose, extensively washed and the radioactivity retained was eluted with 1 mM GTP showing that: (a) radioactivity corresponds to free 125I-VIP and (b) alpha s (Mr 42,000) and beta (Mr 35,000) subunits of Gs protein are detectable in the GTP eluate by immunoblotting using antisera against these subunits. Such an effect of GTP implied that a stable ternary complex consisting of VIP, receptor and Gs protein had been adsorbed to WGA-Sepharose. When Triton-solubilized 125I-VIP-receptor complexes were adsorbed on WGA-Sepharose, then retained material was specifically eluted with 0.3 M N-acetylglucosamine, analysis of the sugar eluate showed the following results. (a) GTP induces the dissociation of 125I-VIP-receptor complexes of Mr 150,000 contained in the eluate indicating that 125I-VIP-receptor-G protein complexes had been adsorbed to the WGA column. (b) The Mr-42,000 alpha s subunit can be specifically ADP-ribosylated by cholera toxin. (c) Immunoblotting using antisera against the alpha s and beta subunits of Gs protein, reveals Mr-42,000 and Mr-35,000 components corresponding to alpha s and beta subunits, respectively. (d) Affinity cross-linking using dithiobis(succinimidyl-propionate) of 125-I-VIP-receptor complexes eluted from the WGA column reveals a major band corresponding to Mr 150,000. Immunoblotting using antisera against the beta-subunit shows the presence of the beta subunit (Mr 35,000) in this Mr-150,000 component. In conclusion, these data provide functional and immunochemical evidence for the physical association of solubilized VIP-receptor complexes with alpha s and beta subunits of Gs protein.     

Pancreatic CCK receptors: Characterization of covalently labeled subunits

¹²⁵I-CCK was crosslinked with ultraviolet light to its receptor on pancreatic plasma membranes. The predominant labeled species following polyacrylamide gel electrophoresis had a molecular weight of 120,000 in the absence, and 80,000 in the presence of the reducing agent dithiothreitol. The M_r = 120,000 labeled band could be extracted, reduced and converted to M_r = 80,000. Moreover, peptide mapping with Staph aureus V8 protease showed a similar pattern for the 120,000 and 80,000 dalton bands. The crosslinked receptor could be solubilized with Triton X-100, absorbed to wheat germ agglutinin and eluted with N-acetylglucosamine. The results indicate, therefore, that the CCK receptor is a glycoprotein with subunits coupled by disulfide bonds.     

Characterization of vasoactive intestinal peptide (VIP) receptors in mammalian lung

125I-VIP bound specifically to sites on human, rat, guinea pig, and rabbit lung membranes with a dissociation constant (KD) of 60-200 pM and binding site maxima of 200-800 fmol/mg of protein. The presence of a second lower affinity site was detected but not investigated further. High affinity 125I-VIP binding was reversible and displaced by structurally related peptides with an order of potency: VIP greater than rGRF greater than PHI greater than hGRF greater than secretin = Ac Tyr1 D Phe2 GRF. 125I-VIP has been covalently incorporated into lung membranes using disuccinimidyl suberate. Sodium dodecyl sulfate-polyacrilamide gel electrophoresis of labeled human, rat, and rabbit lung membranes revealed major 125I-VIP-receptor complexes of: Mr = 65,000, 56,000, and 64,000 daltons, respectively. Guinea pig lung membranes exhibited two 125I-VIP-receptor complexes of Mr = 66,000 and 60,000 daltons. This labeling pattern probably reflects the presence of differentially glycosylated forms of the same receptor since treatment with neuroaminidase resulted in a single homogeneous band (Mr = 57,000 daltons). Soluble covalently labeled VIP receptors from guinea pig and human lung bound to and were specifically eluted from agarose-linked wheat germ agglutinin columns. Our studies indicate that mammalian lung VIP receptors are glycoproteins containing terminal sialic acid residues.     

Multimeric structure of the tumor necrosis factor receptor of HeLa cells

The tumor necrosis factor (TNF) receptor of HeLa cells was solubilized in Triton X-100 and characterized by gel filtration, affinity labeling, and ligand blotting studies. Receptors solubilized with Triton X-100 eluted in gel filtration as a major peak of Mr = 330,000 and retained high affinity binding (KD = 0.25 nM). Affinity labeling of soluble receptor/125I-TNF complexes using the reversible, bifunctional bis[2-(succinimidooxycarbonyl-oxy)ethyl] sulfone resulted in the formation of cross-linked species of Mr = 310,000, 150,000-175,000, 95,000, and 75,000. The formation of these complexes was competitively inhibited by unlabeled TNF. Partial reversal of cross-linking in these complexes and their analysis by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) resolved 125I-TNF dimers cleaved from the 95,000 band and 125I-TNF monomer cleaved from the 75,000 band, providing evidence for a Mr approximately 60,000 subunit. In addition, the 95,000 and 75,000 bands were resolved as components of larger complexes (Mr = 150,000-175,000), which presumably contain two receptor subunits. The Mr 95,000 and 75,000 bands were also released from the Mr 310,000 complex by reduction with dithiothreitol, suggesting a role for disulfide bond stabilization. To investigate the association of the putative receptor subunits, Triton X-100 extracts from HeLa membranes were fractionated by SDS-PAGE without reduction and transferred electrophoretically to nylon membranes for TNF binding assays. Only two bands of Mr = 60,000 and 70,000 specifically bound TNF, and higher Mr binding activity was not observed. These results indicate that TNF receptors in HeLa cells are high molecular weight complexes containing Mr = 60,000 and 70,000 subunits each capable of binding TNF and that the complexes are primarily stabilized by non-covalent, hydrophobic interactions.     

Structural differences between insulin receptors in the brain and peripheral target tissues

Insulin receptors in various brain regions (olfactory tubercle, hippocampus, and hypothalamus) were photoaffinity labeled using the photoreactive analogue of insulin B2(2-nitro,4-azidophenylacetyl)-des-PheB1-insulin (NAPA-DP-insulin). A protein with an apparent Mr of 400,000 was specifically labeled with 125I-NAPA-DP-insulin in all three brain regions. When radiolabeled proteins were reduced with dithiothreitol prior to electrophoresis, specific labeling occurred predominantly in a protein with an apparent Mr of 115,000 and to a much lesser extent in a protein with an apparent Mr of 83,000. The size of these receptor proteins, based on their electrophoretic mobilities, was consistently smaller than insulin receptor proteins in adipocytes. The covalent labeling of insulin receptors in brain by 125I-NAPA-DP-insulin was not blocked by anti-insulin receptor antiserum. Additionally, in contrast to effects observed in peripheral target tissues, this antisera did not inhibit the binding of 125I-insulin to brain membranes. Neuraminidase treatment resulted in an increase in the electrophoretic mobilities of insulin receptor subunits in adipocytes, but, had no effect on receptor subunits in brain. Solubilized insulin receptors from adipocytes were retained by wheat germ agglutinin columns and specifically eluted with N-acetylglucosamine. In contrast, solubilized insulin receptors from brain did not bind to these columns. The results from this study indicate that structural differences, including molecular weight, antigenicity, and carbohydrate composition exist between insulin receptors in brain and peripheral target tissues.     

Affinity labeling of multiplication stimulating activity receptors in membranes from rat and human tissues

Plasma membranes from rat adipocytes and liver and from human placenta have been labeled by covalent cross-linking to membrane-bound 125I-labeled multiplication stimulating activity (125I-MSA) with three different bishydroxysuccinimide esters: disuccinimidyl suberate, disuccinimidyl succinate, and ethyleneglycolyl bis(succinimidyl succinate). Dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiographic analysis of the 125I-MSA-labeled material in the presence of dithiothreitol reveals one single-labeled protein migrating with an apparent Mr = 255,000 regardless of the kind and concentration of cross-linker used. Electrophoresis in the absence of reductant indicates that the affinity-labeled species is not disulfide-linked to any other protein in the native plasma membrane, but contains internal disulfide bonds that compact its structure. The labeling of the Mr = 255,000 species increases with increasing concentrations of 125I-MSA between 0.3 and 3 nM. Labeling is abolished in a competitive manner by nonradioactive MSA but not by similar concentrations of insulin, proinsulin, or epidermal growth factor in all three tissues examined. The unique labeling of this Mr = 225,000 membrane component and its selective inhibition by MSA suggest that this protein is a plasma membrane receptor for MSA.     

Solubilization of somatostatin receptors in hamster pancreatic beta cells. Characterization as a glycoprotein interacting with a GTP-binding protein

Somatostatin receptors of plasma membranes from beta cells of hamster insulinoma were covalently labelled with 125I-[Leu8,D-Trp22,Tyr25]somatostatin-28 (125I-somatostatin-28) and solubilized with the non-denaturing detergent Triton X-100. Analysis by SDS/PAGE and autoradiography revealed three specific 125I-somatostatin-28 receptor complexes with similar molecular masses (228 kDa, 128 kDa and 45 kDa) to those previously identified [Cotroneo, P., Marie, J.-C. & Rosselin, G. (1988) Eur. J. Biochem. 174, 219-224]. The major labelled complex (128 kDa) was adsorbed to a wheat-germ-agglutinin agarose column and eluted by N-acetylglucosamine. Also, the binding of 125I-somatostatin-28 to plasma membranes was specifically inhibited by the GTP analog, guanosine-5'-O-(3-thiotriphosphate) (GTP[S]) in a dose-dependent manner. Furthermore, when somatostatin-28 receptors were solubilized by Triton X-100 as a reversible complex with 125I-somatostatin-28, GTP[S] specifically dissociated the bound ligand to a larger extent from the soluble receptors than from the plasma-membrane-embedded receptors, the radioactivity remaining bound after 15 min at 37 degrees C being 30% and 83% respectively. After pertussis-toxin-induced [32P]ADP-ribosylation of pancreatic membranes, a 41-kDa [32P]ADP-ribose-labelled inhibitory guanine nucleotide binding protein coeluted with the 128-kDa and 45-kDa receptor complexes. The labelling of both receptor proteins was sensitive to GTP[S]. The labelling of the 228-kDa band was inconsistent. These results support the conclusion that beta cell somatostatin receptors can be solubilized as proteins of 128 kDa and 45 kDa. The major labeled species corresponds to the 128-kDa band and is a glycoprotein. The pancreatic membrane contains a 41-kDa GTP-binding protein that can complex with somatostatin receptors.     

The properties of proteoglycan prepared from human articular cartilage by using associative caesium chloride gradients of high and low starting densities.

Reduction of human thyroid membranes with dithiothreitol caused the release of a water-soluble glycoprotein which neutralized the thyrotropin (TSH) receptor-binding and thyroid-stimulating activities of Graves' serum. Analysis of the protein by gel filtration and sucrose density gradient centrifugation allowed estimates of 3.45 nm for the Stokes' radius, 3.6 S for the s20,w and 47 000 +/- 5000 (mean +/- S.D.; n = 4) for the Mr. The material released by dithiothreitol treatment could be crosslinked to 125I-labelled TSH coupled to N-hydroxysuccinimidyl 4-azidobenzoate (125I-HSAB-TSH), suggesting that it contained a component of the TSH receptor. Furthermore, analysis of the crosslinked material by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that it contained the TSH receptor A subunit (Mr 50 000). Several factors suggested therefore that the glycoprotein released by dithiothreitol treatment of human thyroid membranes was the TSH receptor A subunit. In particular, (a) both preparations were hydrophilic and were released from membranes by reduction, (b) they had similar Mr values and (c) both preparations crosslinked to 125I-HSAB-TSH. Material similar to the TSH receptor A subunit was released from thyroid membranes by treatment with papain, probably as a result of cleavage of the receptor A subunit at a site close to the interchain disulphide bridge. A similar mechanism, involving thyroid proteinases, was probably involved in release of material with similar properties to the TSH receptor A subunit during freezing and thawing of human thyroid homogenates.     

Use of the heterobifunctional cross-linker m-maleimidobenzoyl N-hydroxysuccinimide ester to affinity label cholecystokinin binding proteins on rat pancreatic plasma membranes

The binding of 125I-cholecystokinin-33 (125I-CCK-33) to its receptors on rat pancreatic membranes was decreased by modification of membrane protein sulfhydryl groups. Sulfhydryl modifying reagents also caused an accelerated release of bound 125I-CCK-33 from its receptor. Because of the presence of an essential sulfhydryl group(s) in CCK receptor binding we studied the application of the heterobifunctional (SH,NH2) cross-linker, m-maleimidobenzoyl N-hydroxysuccinimide ester (MBS), to affinity label 125I-CCK-33 binding proteins on rat pancreatic plasma membranes. Analysis of the cross-linked products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that this heterobifunctional cross-linker affinity labeled a major Mr = 80,000-95,000 protein previously identified as part of the CCK receptor on the basis of affinity labeling using homobifunctional and heterobifunctional photoreactive cross-linkers. Additional proteins of Mr greater than 200,000, and Mr = 130,000-140,000 were affinity labeled using MBS. The efficiency of the cross-linking reaction between 125I-CCK-33 and its membrane binding proteins with MBS was significantly greater than that obtained with NH2-directed homobifunctional reagents such as disuccinimidyl suberate. The efficiency of cross-linking could be dramatically improved by reduction of membrane proteins with low-molecular weight thiols prior to binding and cross-linking. The differential labeling patterns of the CCK binding proteins obtained with chemical cross-linkers of similar length but different chemical reactivity underscores the need for caution in predicting native receptor structure from affinity labeling data alone. Using the same pancreatic plasma membrane preparation and 125I-insulin, the Mr = 125,000 alpha-subunit of the insulin receptor was affinity labeled using MBS as cross-linker, demonstrating its utility in identifying other peptide hormone receptors.