Intrinsic photoaffinity labeling probes for cholecystokinin (CCK)-gastrin family receptors. D-Tyr-Gly-[Nle28,31,pNO2-Phe33)CCK-26-33)

Attempts to biochemically characterize the pancreatic cholecystokinin (CCK) receptor by affinity labeling have utilized either 125I-Bolton-Hunter-CCK-33 ("long" probes) or decapeptide analogues of the carboxyl terminus of CCK ("short" probes), and covalent attachment via the amino-terminal regions of these probes. The long probe has identified a protein of Mr = 80,000 while "shorter" probes, which have their site of cross-linking closer to the receptor binding region of the probes, have labeled a distinct protein of Mr = 85,000-95,000. To extend and complement these observations, we have designed and synthesized a new probe for the CCK receptor which incorporates a photolabile p-nitrophenylalanine moiety within the theoretical receptor-binding region of the hormone, as its carboxyl-terminal residue. This "intrinsic" photoaffinity labeling probe has been shown to possess full biological activity, with potency and efficacy in stimulating amylase secretion by dispersed rat pancreatic acini similar to that of CCK-8 (CCK-26-33). When iodinated oxidatively, this probe binds rapidly, in a temperature-dependent, reversible, saturable, specific, high affinity manner to enriched pancreatic plasma membranes. In this work, we have used this probe to specifically label the CCK binding site on rat pancreatic plasma membranes. The Mr = 85,000-95,000 protein previously identified with amino-terminal cross-linking of short probes appears to be the protein labeled with this reagent as well. This provides strong evidence that this pancreatic plasma membrane protein contains the CCK-binding domain of the CCK receptor. This intrinsic photoaffinity labeling probe should be quite useful for the characterization of the active site of this receptor and for other CCK and gastrin receptors in many species.     

Use of a nitrotryptophan-containing peptide for photoaffinity labeling the pancreatic cholecystokinin receptor

We report the preparation and characterization of a new type of intrinsic photoaffinity labeling probe, on the basis of the incorporation of a photolabile nitrotryptophan into a biologically relevant domain of a peptide. The model system used was the pancreatic cholecystokinin (CCK) receptor, previously affinity labeled with a variety of probes. Those studies have suggested that an Mr = 85,000-95,000 protein is more likely to be labeled as the site of covalent attachment approaches the receptor-binding domain of this hormone. Indeed, CCK has a Trp in the center of its receptor-binding region, and replacement of that residue with 6-nitrotryptophan resulted in a photolabile probe which affinity labeled the same Mr = 85,000-95,000 pancreatic membrane protein. This probe, 125I-D-Tyr-Gly-[(Nle28,31,6-NO2-Trp30)CCK-26-33], was synthesized by solid-phase and solution techniques and characterized by mass spectrometry. Following oxidative iodination, it was purified on HPLC to 2000 Ci/mmol. Binding to pancreatic membranes was rapid, temperature dependent, reversible, saturable, and specific and was with high affinity (Kd = 3 nM). While its binding affinity was only 3-fold lower than that of native CCK-8, this probe was 70-fold less potent than native hormone in stimulating amylase secretion (EC50 = 1 nM) and equally efficacious to native hormone. Despite the slight decrease in affinity, this probe demonstrated a high relative efficiency of covalent labeling of the Mr = 85,000-95,000 protein. This confirms that the Mr = 85,000-95,000 protein represents the hormone-binding subunit of the CCK receptor and demonstrates the utility of this type of photoaffinity labeling probe.(ABSTRACT TRUNCATED AT 250 WORDS)     

Establishment of a new short, protease-resistant, affinity labeling reagent for the cholecystokinin receptor

Proteolytic degradation of radioligands is an important source of artifact in affinity labeling of receptor proteins. To complement our previous characterization of the pancreatic acinar cell cholecystokinin (CCK) receptor, we synthesized D-Tyr-Gly[(Nle28,31)CCK-26-33]. The amino terminal D-enantiomer of tyrosine provided a site for oxidative iodination, a free amino group for cross-linking, and rendered the peptide resistant to aminopeptidases. The decapeptide was oxidatively iodinated and purified by reverse-phase HPLC to 2,000 Ci/mmol, to yield a probe which was equal in potency and efficacy to CCK-8, and which bound to rat pancreatic membranes in a rapid, reversible, temperature-dependent, specific, saturable and high affinity manner. This probe was resistant to aminopeptidase degradation, and maintained its ability to bind to receptor after incubation with pancreatic membranes or dispersed cells. Affinity labeling of pancreatic membranes with this analogue identified an Mr = 85,000-95,000 molecule. This analogue offers several advantages over existing probes and should be useful for future studies of this and other CCK receptors.     

Analysis of the carbohydrate composition of the pancreatic plasmalemmal glycoprotein affinity labeled by short probes for the cholecystokinin receptor

Affinity labeling of the rat pancreatic cholecystokinin (CCK) receptor with decapeptide probes has identified an Mr = 85,000-95,000 protein, distinct from the Mr = 80,000 component previously labeled with 125I-Bolton Hunter-CCK-33. We have characterized the carbohydrate composition of this novel protein labeled with 125I-D-Tyr-Gly-[(Nle28,31)-CCK-26-33] and disuccinimidyl suberate by using chemical and enzymatic deglycosylation and lectin chromatography. The Mr = 85,000-95,000 component was demonstrated to be an N-linked sialoglycoprotein based on neuraminidase digestion to Mr = 75,000-85,000 and endo-beta-N-acetylglucosaminidase F (Endo F) digestion to Mr = 42,000. This was distinct from the Mr = 65,000 product of Endo F digestion of the protein labeled with 125I-Bolton Hunter-CCK-33. Lack of an effect of endo-beta-N-acetylglucosaminidase H demonstrated the absence of N-linked simple oligosaccharides, while products of chemical deglycosylation with hydrogen fluoride and endo-alpha-N-acetylgalactosaminidase supported the absence of O-linked carbohydrate. The presence of at least four oligosaccharide chains on the core protein was suggested by Endo F digestion of the Mr = 85,000-95,000 protein using limiting enzyme conditions. This glycoprotein was retained on wheat germ agglutininagarose and eluted by N,N',N"-triacetylchitotriose. Identification of the Mr = 85,000-95,000 component on the ectodomain of the plasmalemma of intact pancreatic acini confirmed this to be the fully processed form of the CCK-binding protein.     

Use of photoaffinity probes containing poly(ethylene glycol) spacers for topographical mapping of the cholecystokinin receptor complex

To further define the structure of the pancreatic cholecystokinin (CCK) receptor and the topographical distance relationships between its subunits, we developed a series of monofunctional photoaffinity probes in which a fixed receptor-binding domain was separated from a photolabile nitrophenylacetamido group by defined lengths of a flexible spacer. The well-characterized CCK receptor radioligand 125I-D-Tyr-Gly-[(Nle28,31)CCK-26-33] provided the receptor-binding component of the probes, while the polymer poly(ethylene glycol) (2, 4, 7, and 10 monomer units long) was used as the spacer. The patterns of affinity labeling of rat pancreatic plasma membranes were examined as a function of spacer length. This ranged from 7.3 to 16.2 A, as calculated by root-mean-square end-to-end distances and validated experimentally by time-resolved fluorescence resonance energy transfer measurements. All probes in the series specifically labeled the Mr = 85,000-95,000 glycoprotein with Mr = 42,000 core, which has been proposed to contain the hormone recognition site. In addition, when the spacer length reached 16.2 A, membrane proteins of Mr = 80,000 and Mr = 40,000 were specifically labeled. The product of endo-beta-N-acetylglucosaminidase F digestion of the Mr = 80,000 protein was Mr = 65,000, similar to a protein previously identified in affinity labeling experiments using a CCK-33-based probe. These observations are consistent with the Mr = 85,000-95,000 pancreatic protein representing the hormone-binding subunit of the CCK receptor, while proteins of Mr = 80,000 and Mr = 40,000 may represent noncovalently associated subunits sited within 16.2 A of the binding domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protease peptide mapping of affinity-labeled rat pancreatic cholecystokinin-binding proteins

Affinity-labeling probes with sites of cross-linking distributed along the ligand have been used to biochemically characterize the pancreatic cholecystokinin (CCK) receptor. Probes with photolabile sites spanning the receptor-binding domain have labeled a Mr = 85,000-95,000 plasma membrane protein, while a probe cross-linked via the amino terminus of CCK-33, far removed from the carboxyl-terminal receptor-binding domain, has labeled a distinct Mr = 80,000 protein. In this work, protease peptide mapping of the pancreatic proteins labeled by each of these probes has been performed to gain insight into the identities of the bands and to define domains of the labeled proteins. Photolabile decapeptide probes with sites of cross-linking at the amino terminus, mid region, and carboxyl terminus of the receptor-binding domain each labeled a Mr = 85,000-95,000 glycoprotein with a Mr = 42,000 core protein and similar Staphylococcus aureus V8 protease peptide maps. This confirms that each probe labels the same binding protein and the same domain of that protein. Serial slices through the broad labeled band were separately deglycosylated and protease-treated, demonstrating a single protein core with differential glycosylation. The CCK-33-based probe, however, labeled predominantly two proteins, one having similar sizes in its native and deglycosylated forms to that labeled by the decapeptide probes and a distinct Mr = 80,000 protein. Of note, the peptide map of the protein believed to be the same as that labeled by the shorter probes was different, suggesting that this probe labeled the binding subunit at a site distinct from that which was labeled by the short probes.     

Identification and localization of cholecystokinin-binding sites on rat pancreatic plasma membranes and acinar cells: a biochemical and autoradiographic study

Using the combined approaches of affinity labeling and light and electron microscopic autoradiography, we investigated the identification and localization of cholecystokinin (CCK)-binding sites on rat pancreatic acinar cells. To define the molecular properties of the CCK-binding site, we incubated rat pancreatic plasma membranes with 125-I-CCK-33 for 15 min at 23 degrees C followed by washing and cross- linking with disuccinimidyl suberate. Specific labeling of a major Mr 85,000 component was revealed as assessed by SDS PAGE under reducing conditions and autoradiography of the dried gels. Components of Mr greater than 200,000, Mr 130,000-140,000, and, Mr 55,000 were labeled under maximal cross-linking conditions. The labeling of all components was specifically inhibited by CCK-8 in a dose-dependent manner (Kd approximately 9 nM). The Mr 85,000 component had identical electrophoretic mobilities under reducing and nonreducing conditions indicating that it likely does not contain intramolecular disulfide bonds. The larger labeled species may be cross-linked oligomers of this binding protein or complexes between it and neighboring polypeptides. For studies on the distribution of CCK-binding sites, pancreatic acini were incubated with 125I-CCK-33 (0.1 nM) in the absence or presence of CCK-8 (1 microM) for 2 or 15 min at 37 degrees C, washed, and fixed in 2% glutaraldehyde. Quantitative autoradiographic analysis indicated that approximately 60% of the total grains were located within +/- 1 HD (1 HD = 100 nm) of the lateral and basal plasmalemma with little or no labeling of the apical plasmalemma. From these data, it was estimated that each acinar cell possesses at least 5,000-10,000 CCK-binding sites on its basolateral plasmalemma. The remaining grains showed no preferential concentration over the cytoplasm or nucleus. Together, these data indicate that CCK interacts with a Mr 85,000 protein located on the basolateral plasmalemma of the pancreatic acinar cell.     

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.     

Preparation of enriched plasma membranes from bovine gallbladder muscularis for characterization of cholecystokinin receptors

A method for the preparation of enriched plasma membranes from bovine gallbladder muscularis was developed, validated, and applied to the characterization of receptors for the gastrointestinal hormone cholecystokinin (CCK) on this target. Binding of radioiodinated CCK ligands to this preparation was rapid, reversible, temperature-dependent, saturable, and specific. Only structurally related peptides inhibited CCK binding, and good correlation existed between relative potencies for binding inhibition and for stimulating gallbladder contraction. Computer analysis of CCK-binding data using a nonlinear model-fitting program best fit a model with a single class of sites, with Kd 756 pm and binding capacity 4.5 +/- 1.3 pmol/mg of protein. This degree of enrichment for plasma membranes was adequate for the initial biochemical characterization of this CCK receptor. Affinity labeling using 125I-Bolton Hunter-CCK-33 and m-maleimidobenzoyl-N-hydroxysuccinimide ester identified proteins with Mr = 70,000-85,000, Mr = 120,000-125,000, and Mr = 200,000. Labeling was inhibited in a concentration-dependent manner, with an IC50 of 1 nM CCK-8, and the electrophoretic mobility of these bands was not different under reducing and nonreducing conditions. The major labeled band of Mr = 70,000-85,000 has a lower apparent Mr than that of the analogous band in pancreas labeled with similar methods, supporting the molecular heterogeneity of CCK receptors on these two target tissues.     

The biochemical characterization of the native pancreatic cholecystokinin receptor using affinity labeling approaches.

Affinity labeling has been a powerful tool for the biochemical characterization of sparse molecules which bind to a ligand probe in a specific, high-affinity manner. The rat pancreatic acinar cell receptor for cholecystokinin (CCK), the major physiologic hormonal stimulant of pancreatic exocrine secretion, has been the target of such investigation. Of interest, affinity-labeling studies have identified two distinct plasma membrane glycoproteins as candidates to represent this receptor. The initial candidate, which was identified using 125I-Bolton Hunter-labeled CCK-33 as probe, migrates on a SDS-polyacrylamide gel as a broad band in the M(r) = 80,000 range. Subsequently, using shorter probes in which the site of covalent attachment was closer to the receptor-binding domain of the probe, a band of M(r) = 85,000-95,000 was specifically labeled. Deglycosylation and protease-peptide mapping demonstrated that these bands represent distinct molecules. Using "intrinsic" probes of the receptor, in which a photolabile residue was sited within the pharmacophoric domain of the ligand, attention was focused on the latter candidate as representing the binding protein. Insight into the relationship between these proteins as they reside in the plasma membrane was contributed by labeling with a "topographical mapping" probe, which incorporates a flexible spacer of variable length between a CCK-like ligand and a photolabile residue. This procedure confirmed that these two minor membrane proteins are spatially associated with each other.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gallbladder CCK receptors: species differences in glycosylation of similar protein cores

Receptors for cholecystokinin (CCK) on gallbladder muscularis smooth muscle have different apparent sizes in man (Mr = 85,000-95,000) and cow (Mr = 70,000-85,000). In this work, these receptors were demonstrated to represent N-linked complex glycoproteins with Mr = 43,000 protein cores, based on lectin-affinity chromatography and the deglycosylation of bands affinity labeled with 125I-D-Tyr-Gly-[(Nle28,31, pNO2-Phe33)CCK-26-33] using neuraminidase, O-glycanase and endoglycosidases H and F. Similarities in the core proteins were further demonstrated by Staphylococcus aureus V8 protease peptide mapping, in which both proteins yielded similar fragment patterns. Thus, gallbladder CCK receptors present in man and cow are both N-linked complex glycoproteins, with different carbohydrate domains and similar protein cores.     

Characterization of cholecystokinin receptors in toad retina

The binding characteristics, structure, and pharmacologic properties of a cholecystokinin binding protein in toad retinal membranes have been studied. In competition binding studies using 125I-CCK-8, toad retinal membranes exhibited a high affinity binding site having a Ki50 of 1.5 nM using CCK-8 as competitive ligand. The relative potencies of CCK-related peptides in inhibiting radioligand binding were caerulein greater than gastrin II approximately equal to CCK-8 approximately equal to CCK-33 greater than CCK-8-DS approximately equal to gastrin I. L-364,718, a potent inhibitor of peripheral CCK receptors, was ineffective at competition binding at concentrations up to 1 microM; dibutyryl cyclic GMP was modestly effective at competing (KD approximately 10 mM). Covalent binding of 125I-CCK-33 to toad retinal membranes using chemical cross-linkers or UV irradiation resulted in the labeling of a major Mr 62,000 protein and the intermittent labeling of minor components of Mr 105,000 and Mr 40,000 as determined by SDS-PAGE and autoradiography. The binding of 125I-CCK-33 to retinal membranes and the concomitant labeling of the Mr 62,000 component was specifically inhibited by CCK-8 (KD approximately 1.5 nM). Reduction of membranes with DTT abolished specific binding of 125I-CCK. SDS-PAGE analysis of affinity cross-linked membranes under non-reducing conditions revealed that the Mr 62,000 protein migrated with an apparently lower molecular weight. These results suggest that the Mr 62,000 CCK binding protein in the toad retina contains an intramolecular disulfide bond(s). The Mr 62,000 protein was retained on a wheat germ agglutinin-agarose column and eluted with N-acetyl D-glucosamine, suggesting the glycoprotein nature of this protein. Digestion of the Mr 62,000 protein with neuraminidase together with O-glycanase resulted in a discrete product of Mr approximately 60,000. These results indicate that the Mr 62,000 protein is a glycoprotein with O-linked oligosaccharide chains. Taken together, these data indicate that the CCK receptor in toad retina has a distinct structure compared to that described in rat pancreas or brain. It will be important to establish whether this difference is reflected in differences in signal transduction mechanisms.     

A new probe for affinity labelling pancreatic cholecystokinin receptor with minor modification of its structure

Biochemical studies on receptors for peptides are most often carried out on affinity-labelled (peptide-receptor) complexes. Necessarily, the assumption is made that a covalent (peptide-receptor) complex behaves as the native receptor. The validity of this assumption is dependent on both the affinity-labelling technique and the resolution of the analytical method used for biochemical characterization. We designed a new affinity-labelling probe in order to minimize structural modifications occurring within the affinity-labelled cholecystokinin (CCK) receptor protein. The probe was 125I-labelled 2-(p-azidosalicylamido)-1,3-dithiopropionate-[Thr28,Ahx31 ]CCK-25-33, (125I-ASD-[Thr28,Ahx31]CCK-25-33), the peptide moiety of which was released from its binding site by reduction. It was obtained by coupling a photoactivable chemical to [Thr28,Ahx31]CCK-25-33 via its N-terminus. The resulting peptide was HPLC purified and radioiodinated in the presence of chloramine T. Binding of 125I-ASD-[Thr28,Ahx31]CCK-25-33 was time- and temperature-dependent and reversible. At 25 degrees C, a steady-state level was reached after 60 min and half-maximal dissociation after 38 min. Binding was inhibited by [Thr28,Ahx31]CCK-25-33 and L-364-718 antagonist with IC50 0.4 nM and 0.9 nM, respectively. Photoaffinity labelling of pancreatic plasma membranes by 125I-ASD-[Thr28,Ahx31]CCK-25-33 identified a glycoprotein of Mr 85,000-100,000 which was retained on immobilized wheat germ agglutinin. Enzyme cleavage by endoproteinase Glu-C generated a main fragment of Mr 30,000-34,000. The same glycoprotein was photoaffinity labelled with 125I-DTyr-Gly-[Ahx28,31,pNO2Phe33]CCK-26-33 (Ahx, 2-aminohexanoic acid; pNO2Phe,p-nitrophenylalanine) an intrinsic probe having its photolabile group sited in the binding domain of cholecystokinin. 125I-ASD-[Thr28,Ahx31]CCK-25-33 is a potentially powerful tool for biologically and biochemically studying cholecystokinin receptors.     

Identification of a receptor for peptides of the bombesin family in Swiss 3T3 cells by affinity cross-linking

The cross-linking agent ethylene glycol-bis(succinimidyl succinate) was used to covalently link 125I-labeled gastrin releasing peptide (125I-GRP) to an Mr 75,000-85,000 surface protein in Swiss 3T3 cells that displays many characteristics of a specific receptor for peptides of the bombesin family. This protein was not present in other cell lines which do not exhibit receptors for bombesin-like peptides. Unlabeled GRP competed for affinity labeling of the Mr 75,000-85,000 protein in a concentration-dependent manner, and other bombesin-related peptides also inhibited the cross-linking of 125I-GRP to this component. In contrast, high concentrations of a variety of other peptide hormones and mitogens had no effect. Affinity labeling of the Mr 75,000-85,000 protein was dependent on the concentration of 125I-GRP and exhibited saturability. 125I-GRP affinity labeling of this protein was also demonstrated by two-dimensional gel electrophoresis. These studies suggest that an Mr 75,000-85,000 surface protein with an isoelectric point of 6.0 to 6.5 is a major component of the receptor for peptides of the bombesin family in Swiss 3T3 cells.     

Analysis of cholecystokinin-binding proteins using endo-beta-N-acetylglucosaminidase F

We have previously shown that the cholecystokinin (CCK)-binding proteins in rat pancreatic plasma membranes consist of a major Mr 85,000 and minor Mr 55,000 and Mr 130,000 species as revealed by affinity labeling with 125I-CCK-33 using the cross-linker, disuccinimidyl suberate. The glycoprotein nature of these species was investigated using endoglycosidase F (endo F) and neuraminidase treatment and wheat germ agglutinin-agarose chromatography. Treatment of affinity-labeled membranes with endo F resulted in increased electrophoretic mobilities of all three binding proteins, indicating removal of N-linked oligosaccharide side chains. Endo F treatment of each protein in gel slices indicated the following cleavage relationships: Mr 85,000----65,000; Mr 55,000----45,000; Mr 130,000---- 110,000. Using limiting enzyme conditions to digest each protein contained in excised SDS gel slices, three and four products, respectively, were identified for the Mr 85,000 and 55,000 proteins. Similar treatment of the Mr 130,000 protein revealed only the Mr 110,000 product. These results indicated that the Mr 85,000 protein has at least three, the Mr 55,000 protein has at least four, and the Mr 130,000 protein has at least one, N-linked oligosaccharide side chain(s) on their polypeptide backbone. Neuraminidase treatment of affinity-labeled membranes caused slight increases in the electrophoretic mobilities of all three proteins, indicating the presence of sialic acid residues. Solubilization of affinity-labeled membranes in Nonidet P-40 followed by affinity chromatography on wheat germ agglutinin-agarose revealed that all three CCK-binding proteins specifically interact with this lectin and can be eluted with N-acetyl- D-glucosamine. Analysis of the proteins present in the eluted fractions by silver staining indicated a significant enrichment for proteins having molecular weights corresponding to the major CCK-binding proteins in comparison to the pattern of native membranes. Taken together, these studies provide definitive evidence that the CCK- binding proteins in rat pancreas are (sialo)glycoproteins.     

Specific photoaffinity crosslinking of [125I]cholecystokinin to pancreatic plasma membranes. Evidence for a disulfide-linked Mr 76 000 peptide in cholecystokinin receptors

In rat pancreatic plasma membranes, preincubated with [125I]cholecystokinin-33 (CCK-33) and washed free of unbound tracer, the irradiation by UV light induced the irreversible binding of radioactivity to high molecular weight peptides as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) and autoradiography. This was not observed when the membranes were preincubated in the simultaneous presence of [125I]CCK-33 and of either an excess of unlabelled CCK-8 or of guanosine 5'-(beta, gamma-imido)-triphosphate. The radioactivity was mostly crosslinked with a Mr 96,000 peptide and peptide species of Mr greater than 200,000, after SDS solubilization in the absence of beta-mercaptoethanol. Peptide reduction with beta-mercaptoethanol converted the high molecular weight radioactive species into a Mr 76,000 peptide that contained as much as 65% of the radioactivity crosslinked. The Mr 76,000 peptide appears, therefore, to be a disulfide-linked constituent of rat pancreatic cholecystokinin receptors.     

Characterization of cholecystokinin receptors in bullfrog (Rana catesbeiana) brain and pancreas

The binding of biologically active 125I-Bolton-Hunter-CCK-33 to bullfrog brain and pancreatic membrane particles was characterized. Both tissues exhibited time-dependent, saturable, reversible, and high affinity binding without evidence for cooperative interaction. Both bullfrog CCK receptors resembled their mammalian counterparts in having acidic pH optima for tracer binding and a Kd of about 0.5 nM. However, the receptors differed from their mammalian counterparts in that (1) the bullfrog brain membranes bound more tracer per mg protein than did the pancreatic membranes, (2) both bullfrog CCK receptors were relatively insensitive to dibutyryl cGMP, and (3) both bullfrog brain and pancreatic CCK receptors exhibited the same general specificity toward a variety of CCK and gastrin peptides. For both tissues, the relative order of receptor binding potency was CCK-8 greater than caerulein = CCK-33 greater than gastrin-17-II greater than CCK-8-ns = gastrin-17-I greater than caerulein-ns greater than gastrin-4 with the sulfated CCK peptides being 1000-fold more potent than their nonsulfated analogs. Sulfated gastrin was also relatively potent, being only 10-fold weaker than CCK-8. Gastrin-4 was 20 000-fold weaker than CCK-8 in interacting with the brain CCK receptor. The latter finding is in sharp contrast to the mammalian brain CCK receptor. We conclude that the bullfrog brain and pancreas contain similar CCK receptors of probable physiological significance and may represent an ancestral condition from which the two distinct CCK receptors present in mammalian brain and pancreas have evolved.     

Purification and characterization of the rat pancreatic cholecystokinin receptor

Cholecystokinin (CCK) is a peptide hormone that has a variety of physiologically important functions in the gastrointestinal tract, in which distinct high affinity receptors have been identified. We describe here the purification of the digitonin-solubilized rat pancreatic receptor as an initial step in the determination of its primary structure. Solubilization of total pancreatic membranes using 1% digitonin resulted in a single class of binding sites with a specific content of 4 pmol/mg as measured in a soluble binding assay using the nonpeptidyl CCK antagonist [3H]3S[-]-N-[2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4- benzodiazepine-3-yl]-1H-indole-2-carboxamide [( 3H]364,718). The solubilized receptor was purified using the following chromatographic steps: 1) cation exchange; 2) Ulex europaeus agglutinin-I-agarose; and 3) Sephacryl S-300. The final preparation of the purified receptor had a specific content of 8,055 pmol/mg, which represented a 9,051-fold purification from intact membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified receptor preparation under reducing conditions resulted in a predominant polypeptide with an Mr = 85,000-95,000 and minor polypeptides of Mr = 57,000 and 26,000 as determined by radiolabeling and silver staining. Solubilized pancreatic membranes were affinity labeled with the peptidyl CCK agonist 125I-D-Tyr-Gly-[(Nle28,31,6-NO2-Phe33)CCK-26-33] and chromatographed under conditions similar to those described for untreated membranes. Elution of radioactive peaks from each chromatographic column was coincident with [3H]364,718 binding activity and resulted in a labeled polypeptide having the same electrophoretic mobility as receptor derived from freshly labeled membranes and purified from untreated membranes. High performance liquid-gel exclusion chromatography of the crude digitonin-solubilized membrane preparation revealed an estimated molecular size for the [3H]364,718-binding activity of 370,000, which was consistent with the size determined by nondenaturing gel electrophoresis of the purified receptor complexed with the labeled nonpeptidyl antagonist. Binding of [3H]364,718 to the purified receptor preparation was comparable to that observed with the crude solubilized pancreatic membrane preparation; and both the homologous ligand 364,718 (Ki = 0.5 nm) and CCK-8 (Ki = 1.4 microM) competed for binding to both preparations in a similar manner.     

Multiple neurotransmitter receptors in the brain: amines, adenosine, and cholecystokinin

Radioligand binding studies of neurotransmitter receptors have provided discrimination at the molecular level, permitting the differentiation of multiple receptor subtypes for several biogenic amines. Using this paradigm we have labeled two distinct receptors each for cholecystokinin (CCK) and for adenosine. Adenosine receptors were labeled in brain with [3H]N6-cyclohexyladenosine (3H-CHA) and [3H]1,3-diethyl-8-phenylxanthine (3H-DP). The adenosine receptor labeled by 3H-CHA appears to be an A1 site, associated with reduction of adenylate cyclase activity, while 3H-DP sites resemble A2 receptors linked to adenylate cyclase enhancement. Cholecystokinin-33 labeled by the Bolton-Hunter procedure with 125I(125I-BH-CCK) labels different receptors in brain and pancreas. The pancreatic receptor does not react with CCK derivatives of fewer than eight amino acids, while the brain receptor does recognize pentagastrin, the carboxyl-terminal five amino acids of CCK. The "brain type" CCK receptor may normally interact with CCK-4, the carboxyl-terminal tetrapeptide of CCK, recently identified as a unique neuropeptide highly concentrated in the brain. CCK-8, the other major molecular form of CCK, may be the endogenous ligand for the "pancreatic type" receptor.     

Purification of the pancreatic cholecystokinin receptor

We have previously shown that the pancreatic cholecystokinin (CCK) receptor can be solubilized in 1% digitonin. In this study, digitonin-solubilized CCK receptors from rat pancreas were purified using sequential affinity chromatography on ricin-II agarose and on AffiGel-CCK. Electrophoresis of the radioiodinated purified receptors on SDS-polyacrylamide gels followed by autoradiography revealed two proteins: a major band of Mr = 80,000-90,000, and a minor band of Mr = 55,000. Through the purification procedure, the receptors preserved their agonist specificity (CCK-8 less than CCK-33 less than desulfated CCK-8 less than CCK-4) and binding affinity. Scatchard transformations of binding data for the purified receptor preparation were best fit by linear plots compatible with a single class of binding sites with Kd = 9.4 nM. The estimated purification was about 80,000 fold and consistent with the expected Bmax for a pure Mr = 80,000 protein binding one CCK molecule. This two-step purification procedure opens the possibility for molecular studies of the CCK receptor.