Screening of soy and milk protein hydrolysates for their ability to activate the CCK1 receptor

The cholecystokinin receptor-type 1 (CCK1R) is a G-protein coupled receptor localized in the animal gastrointestinal tract. Receptor activation by the natural peptide ligand CCK leads to a feeling of satiety. In this study, hydrolysates from soy and milk proteins were evaluated for their potential to activate the CCK1R, assuming that bioactive peptides with a satiogenic effect can be used as an effective therapeutic strategy for obesity. Different protein hydrolysates were screened with a cell-based bioassay, which relies on the generation of a fluorescent signal upon receptor activation. Fluorescence was monitored using a fluorescence plate reader and confocal microscopy. Results from the fluorescence plate reader were biased by background autofluorescence of the protein hydrolysate matrices, which makes the fluorescence plate reader inappropriate for the evaluation of complex formulations. Measurements with the confocal microscope resulted in reliable and specific results. The latter approach showed that the gastrointestinal digested 7S fraction of soy protein demonstrates CCK1R activity.     

Miniaturization of intracellular calcium functional assays to 1536-well plate format using a fluorometric imaging plate reader

The measurement of intracellular calcium response transients in living mammalian cells is a popular functional assay for identification of agonists and antagonists to receptors or channels of pharmacological interest. In recent years, advances in fluorescence-based detection techniques and automation technologies have facilitated the adaptation of this assay to 384-well microplate format high-throughput screening (HTS) assays. However, the cost and time required performing the intracellular calcium HTS assays in the 384-well format can be prohibitive for HTS campaigns of greater than 1 x 10(6) wells. For these reasons, it is attractive to miniaturize intracellular calcium functional assays to the 1536-well microplate format, where assay volumes and plate throughput can be decreased by several fold. The focus of the research described in this article is the miniaturization of an intracellular calcium assay to 1536-well plate format. This was accomplished by modifying the hardware and software of a fluorometric imaging plate reader (FLIPR) to enable transfer of nanoliters of test compound directly to a 1536-well assay plate, and measure the resulting calcium response from all 1536 wells simultaneously. An intracellular calcium functional assay against the rat muscarinic acetylcholine receptor subtype 1 (rmAchR1) G-protein coupled receptor (GPCR) was miniaturized and executed on this modified instrument. In experiments measuring the activity of known muscarinic receptor agonists and antagonists, the miniaturized FLIPR assay gave EC(50) and IC(50) values and rank order potency comparable to the 384-well format assays. Calculated Z' factors for the miniaturized agonist and antagonist assays were, respectively, 0.56 +/- 0.21 and 0.53 +/- 0.22, which were slightly higher (Z'(agonist) = 0.55 +/- 0.33) and lower (Z'(antagonist) = 0.70 +/- 0.18) than the corresponding values in the 384-well assays. A mock agonist HTS campaign against the muscarinic receptor in miniaturized format was able to identify all wells spiked with the rmAchR1 agonist carbachol.     

Evaluation of no-wash calcium assay kits: enabling tools for calcium mobilization

The no-wash calcium assay kits developed by Molecular Devices Corporation have greatly enhanced the throughput of cell-based calcium mobilization high-throughput screening (HTS) assays and enabled screening using nonadherent cells. The fluorescent imaging plate reader (FLIPR) Calcium 3 Assay Kit, optimal for targets that have proteins or peptides as agonists, has 2 potential drawbacks: 1) a significant downward spike in fluorescence signal upon liquid transfer that can be the same magnitude as the agonist response, making data analysis difficult; and 2) medium removal is required for some targets, which essentially reintroduces a wash step. Several no-wash products were introduced in 2005. The authors compare the Fluo-4 NW Calcium Assay Kit and the BD Calcium Assay Kit with the FLIPR Calcium 3 Assay Kit using human native rhabdomyosarcoma cells expressing the urotensin-II receptor (UT). The BDtrade mark Calcium Assay Kit gives the best performance in the true no-wash mode, in which both agonist and antagonist activity are easily quantified. Although these new products provide additional options for measuring calcium mobilization, the different results observed with each kit, using the UT receptor as an example, suggest that one should characterize all dyes against each target in a systematic way prior to choosing one for HTS.     

Effects of cholecystokinin-58 on type 1 cholecystokinin receptor function and regulation

Cholecystokinin, like many peptide hormones, is present as multiple molecular forms. CCK-58 has been identified as the dominant form in the circulation, whereas most of the studies of CCK-receptor interactions have been performed with CCK-8. Despite both sharing the pharmacophoric region of CCK, representing its carboxy terminal heptapeptide amide, studies in vivo have demonstrated biological diversity of action of the two peptides, with CCK-58, but not CCK-8, stimulating pancreatic fluid secretion and lengthening the interval between meals. Here, we have directly studied the ability of these two CCK peptides to bind to the type 1 CCK receptor and to stimulate it to elicit an intracellular calcium response. The calcium response relative to receptor occupation was identical for CCK-58 and CCK-8, with the longer peptide binding with approximately fivefold lower affinity. We also examined the ability of the two peptides to elicit receptor internalization using morphological techniques and to disrupt the constitutive oligomerization of the CCK receptor using receptor bioluminescence resonance energy transfer. Here, both full agonist peptides had similar effects on these regulatory processes. These data suggest that both molecular forms of CCK act at the CCK1 receptor quite similarly and elicit similar regulatory processes for that receptor, suggesting that the differences in biological activity observed in vivo most likely reflect differences in the clearance and/or metabolism of these long and short forms of CCK peptides.     

Molecular Basis for Benzodiazepine Agonist Action at the Type 1 Cholecystokinin Receptor

Understanding the molecular basis of drug action can facilitate development of more potent and selective drugs. Here, we explore the molecular basis for action of a unique small molecule ligand that is a type 1 cholecystokinin (CCK) receptor agonist and type 2 CCK receptor antagonist, GI181771X. We characterize its binding utilizing structurally related radioiodinated ligands selective for CCK receptor subtypes that utilize the same allosteric ligand-binding pocket, using wild-type receptors and chimeric constructs exchanging the distinct residues lining this pocket. Intracellular calcium assays were performed to determine biological activity. Molecular models for docking small molecule agonists to the type 1 CCK receptor were developed using a ligand-guided refinement approach. The optimal model was distinct from the previous antagonist model for the same receptor and was mechanistically consistent with the current mutagenesis data. This study revealed a key role for Leu^7.39 that was predicted to interact with the isopropyl group in the N1 position of the benzodiazepine that acts as a “trigger” for biological activity. The molecular model was predictive of binding of other small molecule agonists, effectively distinguishing these from 1065 approved drug decoys with an area under curve value of 99%. The model also selectively enriched for agonist compounds, with 130 agonists identified by ROC analysis when seeded in 2175 non-agonist ligands of the type 1 CCK receptor (area under curve 78%). Benzodiazepine agonists in this series docked in consistent pose within this pocket, with a key role played by Leu^7.39, whereas the role of this residue was less clear for chemically distinct agonists.     

Differential effects of modification of membrane cholesterol and sphingolipids on the conformation, function, and trafficking of the G protein-coupled cholecystokinin receptor

The lipid microenvironment of receptors can influence their conformation, function, and regulation. Cholecystokinin (CCK)-stimulated signaling is abnormal in some forms of hyperlipidemia, suggesting the possibility of unique sensitivity to its lipid environment. Here we examined the influence of cholesterol and sphingolipids on CCK receptors in model Chinese hamster ovary cell systems having lipid levels modified. Cholesterol was modulated chemically or metabolically, and sphingolipids were modulated using a temperature-sensitive cell line (SPB-1). Receptor conformation was probed with a fluorescent full agonist ligand, Alexa 488-conjugated Gly-[Nle(28,31)]CCK-(26-33), shown previously to decrease in anisotropy and lifetime when occupying a receptor in the active conformation (Harikumar, K. G., Pinon, D. L., Wessels, W. S., Prendergast, F. G., and Miller, L. J. (2002) J. Biol. Chem. 277, 18552-18560). Anisotropy and lifetime of this probe were increased and prolonged with cholesterol enrichment, and decreased and shortened with depletion of cholesterol or sphingolipids. The increase in these parameters with cholesterol enrichment may reflect change in CCK receptor conformation toward its inactive, uncoupled state. Indeed, cholesterol enrichment resulted in nonproductive agonist ligand binding, with affinity of binding higher than normal and calcium signaling in response to this reduced. In cholesterol- and sphingolipid-depleted states, the receptor moved into conformations that were less than optimal. With cholesterol depletion, both ligand binding and signaling were decreased, yet internalization and trafficking were unperturbed. With sphingolipid depletion, ligand binding and signaling were normal, but internalization and trafficking were markedly inhibited. Of note, normal transferrin receptor trafficking through the same clathrin-dependent pathway was maintained under these conditions. Thus, lipid microenvironment of the CCK receptor is particularly important, with different lipids having distinct effects.     

Agonist-dependent dissociation of oligomeric complexes of G protein-coupled cholecystokinin receptors demonstrated in living cells using bioluminescence resonance energy transfer.

Dimerization of some G protein-coupled receptors has recently been demonstrated, but how widespread this phenomenon might be and its functional implications are not yet clear. We have utilized biophysical and biochemical techniques to evaluate whether the type A cholecystokinin (CCK) receptor can form oligomeric complexes in the plasma membrane and the impact of ligand binding and signaling on such complexes. We investigated the possibility of bioluminescence resonance energy transfer (BRET) between receptor constructs that included carboxyl-terminal tags of Renilla luciferase or yellow fluorescent protein. Indeed, co-expression of these constructs in COS cells resulted in the constitutive presence of a significant BRET signal above that in a series of controls, with this signal reduced by co-expression of competing non-tagged CCK receptors. The presence of an oligomeric complex of CCK receptor molecules was confirmed in co-immunoprecipitation experiments. Occupation of CCK receptors with agonist ligands (CCK or gastrin-4) resulted in the rapid reduction in BRET signal in contrast to the enhancement of such a signal reported after agonist occupation of the beta(2)-adrenergic receptor. These effects on CCK receptor oligomerization were concentration-dependent, correlating with the potencies of the agonists. A smaller effect was observed for a partial agonist, and no effect was observed for antagonist occupation of this receptor. Agonist-induced reduction in BRET signal was also observed for pairs of CCK receptors with a donor-acceptor pair situated in other positions within the receptor. Manipulation of the phosphorylation state of CCK receptor using protein kinase C activation with phorbol ester or inhibition with staurosporine had no effect on the basal level or agonist effect on CCK receptor oligomerization. This provides the first evidence for CCK receptor oligomerization in living cells, with insights that the active conformation of this receptor dissociates these complexes in a phosphorylation-independent manner.     

Regulation of membrane cholecystokinin-2 receptor by agonists enables classification of partial agonists as biased agonists

Given the importance of G-protein-coupled receptors as pharmacological targets in medicine, efforts directed at understanding the molecular mechanism by which pharmacological compounds regulate their presence at the cell surface is of paramount importance. In this context, using confocal microscopy and bioluminescence resonance energy transfer, we have investigated internalization and intracellular trafficking of the cholecystokinin-2 receptor (CCK2R) in response to both natural and synthetic ligands with different pharmacological features. We found that CCK and gastrin, which are full agonists on CCK2R-induced inositol phosphate production, rapidly and abundantly stimulate internalization. Internalized CCK2R did not rapidly recycle to plasma membrane but instead was directed to late endosomes/lysosomes. CCK2R endocytosis involves clathrin-coated pits and dynamin and high affinity and prolonged binding of β-arrestin1 or -2. Partial agonists and antagonists on CCK2R-induced inositol phosphate formation and ERK1/2 phosphorylation did not stimulate CCK2R internalization or β-arrestin recruitment to the CCK2R but blocked full agonist-induced internalization and β-arrestin recruitment. The extreme C-terminal region of the CCK2R (and more precisely phosphorylatable residues Ser(437)-Xaa(438)-Thr(439)-Thr(440)-Xaa(441)-Ser(442)-Thr(443)) were critical for β-arrestin recruitment. However, this region and β-arrestins were dispensable for CCK2R internalization. In conclusion, this study allowed us to classify the human CCK2R as a member of class B G-protein-coupled receptors with regard to its endocytosis features and identified biased agonists of the CCK2R. These new important insights will allow us to investigate the role of internalized CCK2R·β-arrestin complexes in cancers expressing this receptor and to develop new diagnosis and therapeutic strategies targeting this receptor.     

Molecular mechanism underlying partial and full agonism mediated by the human cholecystokinin-1 receptor

The cholecystokinin-1 receptor (CCK1R) is a G protein-coupled receptor (GPCR) that regulates important physiological functions. As for other GPCRs, the molecular basis of full and partial agonism is still far from clearly understood. In the present report, using both laboratory experiments and molecular modeling approaches, we have investigated the partial agonism mechanism of JMV 180, on the human CCK1R. We first showed that efficacy of the CCK1R to activate phospholipase C is dependent on the correct orientation of the C-terminal end of peptidic ligands toward residue Phe(330) of helix VI. We have previously reported that a single mutation of Met(121) (helix III) markedly reduced the receptor-mediated inositol phosphate production upon stimulation by CCK. Computational simulations predicted that residue 121 affected orientation of the C-terminal end of CCK, thus suggesting that the molecular complex with a reduced inositol phosphate production observed with the mutated CCK1R resembles that resulting from binding of JMV 180 to the WT-CCK1R. Pharmacological, biochemical, and functional characterizations of the two receptor.ligand complexes with decreased abilities to signal were carried out in different cell types. We found that they presented the same features, such as total dependence of inositol phosphate production to Galpha(q) expression, single affinity of binding sites, insensitivity of binding to non-hydrolyzable GTP, absence of GTPgamma[S(35)] binding following agonist stimulation, similarity of dose-response curves for amylase secretion, and incapacity to induce acute pancreatitis in pancreatic acini. We concluded that helices VI and III of the CCK1R are functionally linked through the CCK1R agonist binding site and that positioning of the C-terminal ends of peptidic agonists toward Phe(330) of helix VI is responsible for extent of phospholipase C activation through Galpha(q) coupling. Given the potential therapeutic interest of partial agonists such as JMV 180, our structural data will serve for target structure-based design of new CCK1R ligands.     

Comparison of 3 AT1 receptor binding assays: filtration assay, ScreenReady Target, and WGA Flashplate

In this article, the study of 3 different angiotensin II type 1 (AT(1)) receptor binding assays in terms of reproducibility, robustness, and feasibility for high-throughput screening (HTS) is described. The following methods were used: a nonhomogeneous filtration assay in a 96-well format using CHO-AT(1) cell membranes and 2 homogeneous assays, which include the commercially available ScreenReady Target for the AT(1) receptor and the wheat germ agglutinin (WGA) Flashplate, which was coated "in-house" with the CHO-AT(1) cell membranes. Receptors were labeled with [(125)I]-Sar(1)-Ile(8)-angiotensin II, and radioligand binding was displaced using the antagonist losartan and the natural agonist angiotensin II. Reproducible K(d), B(max), and K(i) values and good total binding/nonspecific binding (TB/NSB) ratios were obtained with both the ScreenReady Targets and the filtration assay, whereas the WGA Flashplates showed unacceptably high nonspecific binding and high variation when applied as a homogeneous assay. However, when applied as a heterogeneous assay (i.e., when a wash step at the end of the assay is included), the results were significantly better. Interestingly, ligand affinities were consistently lower in Flashplate-based assays than in the filtration assay. This may be due to the immobilization of the receptors onto the solid surface of the plate, affecting their conformation. In terms of reproducibility, robustness, and feasibility for HTS, the authors conclude that the ScreenReady Target plates are most suitable for AT(1) receptor binding screening.     

Discovery of novel P2Y14 agonist and antagonist using conventional and nonconventional methods

P2Y14 is a member of the pyrimidinergic GPCR family. UDP-Glc has been previously shown to activate human P2Y14, whereas UDP was unable to activate the receptor. In this study, the authors used conventional and nonconventional methods to further characterize P2Y14 and its ligands. Conventional calcium mobilization and nonconventional cellular impedance functional assays revealed that UMP and UDP selectively activated HEK cells coexpressing P2Y14 and Gα(qi5). In the impedance assays, the presence of exogenous Gα(qi5) resulted in agonist-induced Gq signaling, whereas in the absence of exogenous Gα(qi5), the signal was indicative of Gi. The authors established the first P2Y14 membrane filtration binding assay using a novel optimized expression vector and [(3)H]UDP as radioligand. UDP-Glc, UMP, and UDP dose dependently inhibited [(3)H]UDP binding in the binding assay, and saturation analysis revealed that UDP bound P2Y14 with a K(D) = 10 nM and a B(max) = 110 pmol/mg. The authors screened a phosphonate library and identified compound A, which inhibited UDP-Glc-mediated calcium signaling in the fluorometric imaging plate reader assay (IC(50) = 2.3 μM) and competed for [(3)H]UDP binding in the novel binding assay with a K(i) = 1280 nM.     

A flow-through fluorescence polarization detection system for measuring GPCR-mediated modulation of cAMP production

A flow-through fluorescence polarization (FP) detection system that makes use of a novel high-performance liquid chromatography (HPLC) fluorescence detector modified with polarization filters was developed. This flow-through FP detection system was evaluated by using a novel and very cost-effective bioassay for cyclic adenosine monophosphate (cAMP). The bioassay was first evaluated and optimized in an FP plate reader format and subsequently in a flow-through bioassay setup. The principle of the bioassay is based on the competition of cAMP and a fluorescent cAMP derivative for the cAMP binding domain of protein kinase A. cAMP could accurately be determined over a range of 0.8 to 30 pmol/well in the plate reader FP assay and over a range of 0.3 to 50 pmol/well in the flow-through FP assay setup. High Z' factors (i.e., 0.89 for the plate reader and 0.93 for the flow-through FP cAMP assay, respectively) indicated robust assays. Finally, functional cAMP signaling of the human histamine H(3) G-protein-coupled receptor (GPCR) in cell cultures was measured with both assay formats with good sensitivities and assay windows. The pEC(50) values obtained in both assay formats were in accordance with those obtained with standard methods. The flow-through FP detection system could thus be used as a cost-effective alternative to FP plate reader assays. Moreover, the novel flow-through FP detection system for cAMP constitutes a good analytical tool to be used in the GPCR research field as an alternative to the use of FP plate readers or radioactive laboratories nowadays used for cAMP measurements.     

Regulation of the human bradykinin B2 receptor expressed in sf21 insect cells: a possible role for tyrosine kinases

The functional regulation of the human bradykinin B2 receptor expressed in sf21 cells was studied. Human bradykinin B2 receptors were immunodetected as a band of 75-80 kDa in membranes from recombinant baculovirus-infected cells and visualized at the plasma membrane, by confocal microscopy, using an antibody against an epitope from its second extracellular loop. B2 receptors, detected in membranes by [(3)H-bradykinin] binding, showed a Kd of 0.66 nmol/L and an expression level of 2.57 pmol/mg of protein at 54 h postinfection. In these cells, bradykinin induced a transient increase of intracellular calcium ([Ca(2+)](i)) in fura 2-AM loaded sf21 cells, and promoted [(35)S]-GTP(gamma)S binding to membranes. The effects of bradykinin were dose dependent (with an EC(50) of 50 nmol/L for calcium mobilization) and were inhibited by N-alpha-adamantaneacetyl-D-Arg-[Hyp(3),Thi(5,8),D-phe(7)]-Bk, a specific B2 receptor antagonist. When the B2 antagonist was applied at the top of the calcium transient, it accelerated the decline of the peak, suggesting that calcium mobilization at this point was still influenced by receptor occupation. No calcium mobilization was elicited by 1 micromol/L (Des-Arg(9))-Bk, a B1 receptor agonist that did not inhibit the subsequent action of 100 nmol/L bradykinin. No effect of bradykinin was detected in uninfected cells or cells infected with the wild-type baculovirus. Bradykinin-induced [Ca(2+)](i) mobilization was increased by genistein and tyrphostin A51. These tyrosine kinase inhibitors did not modify basal levels of [Ca(2+)](i). Homologous desensitization of the B2 receptor was observed after repeated applications of bradykinin, which resulted in attenuated changes in intracellular calcium. In addition, genistein promoted an increased response to a third exposure to the agonist when applied after washing the cells that had been previously challenged with two increasing doses of bradykinin. Genistein did not affect the calcium mobilization induced by activation of the endogenous octopamine G protein-coupled receptor or by thapsigargin. The B2 receptor, detected by confocal microscopy in unpermeabilized cells, remained constant at the surface of cells stimulated with bradykinin for 10 min, in the presence or absence of genistein. Agonist-promoted phosphorylation of the B2 receptor was markedly accentuated by genistein treatment. Phosphoaminoacid analysis revealed the presence of phosphoserine and traces of phosphothreonine, but not phosphotyrosine, suggesting that the putative tyrosine kinase(s), activated by bradykinin, could act in a step previous to receptor phosphorylation. Interestingly, genistein prevented agonist-induced G protein uncoupling from B2 receptors, determined by in vitro bradykinin-stimulated [(35)S]-GTP(gamma)S binding, in membranes from bradykinin pretreated cells. Our results suggest that tyrosine kinase(s) regulate the activity of the human B2 receptor in sf21 cells by affecting its coupling to G proteins and its phosphorylation.     

Determination of affinity and activity of ligands at the human neuropeptide Y Y4 receptor by flow cytometry and aequorin luminescence

Fluorescence-labeled neuropeptide Y (NPY) has been used in flow cytometric binding assays for the determination of affinity constants of NPY Y1, Y2, and Y5 receptor ligands. Because the binding of fluorescent NPY is insufficient for competition studies at the human Y4 receptor (hY4R), we replaced Glu-4 in hPP with Lys for the derivatization with cyanine-5. Because cy5-[K(4)]hPP has high affinity (Kd 5.6 nM) to the hY4R, it was used as a probe in a flow cytometric binding assay. Specific binding of cy5-[K(4)]hPP to hY4R was visualized by confocal microscopy. The hY(4)R, the chimeric G protein G(qi5) and mitochondrially targeted apoaequorin were stably coexpressed in CHO cells. Aequorin luminescence was quantified in a microplate reader and by a CCD camera. By application of these methods 3-cyclohexyl-N-[(3-1H-imidazol-4-ylpropylamino)(imino)methyl]propanamide (UR-AK49) was discovered as the first nonpeptidic Y4R antagonist (pKi 4.17), a lead to be optimized in terms of potency and selectivity.     

Ligand-regulated internalization and recycling of human beta 2-adrenergic receptors between the plasma membrane and endosomes containing transferrin receptors.

Agonist-regulated redistribution of human beta 2-adrenergic receptors was examined in 293 cells. A specific antiserum recognizing the carboxyl-terminal hydrophilic domain of the receptor was developed, characterized, and used for immunocytochemical localization of receptors in fixed cells by conventional fluorescence and confocal fluorescence microscopy. The beta-adrenergic agonist isoproterenol induced redistribution of receptors from the surface of cells into small (less than 1 micron diameter) punctuate accumulations which were detected in cells within 2 min of agonist addition. The time course of receptor redistribution paralleled that of receptor sequestration measured by ligand binding, and receptor redistribution was reversible in the presence of the beta-adrenergic antagonist alprenolol. Optical sections imaged through cells by confocal microscopy localized receptor accumulations within the cytoplasm. To address the question of receptor internalization further, a mutant receptor possessing an engineered antigenic epitope in the amino-terminal hydrophilic domain was constructed, transfected into cells, and localized using both a monoclonal antibody recognizing the epitope tag (receptor ectodomain) and an antiserum recognizing the carboxyl terminus (receptor endodomain). In untreated cells most receptor antigen was detected at the cell surface, as assessed by accessibility to ectodomain antibodies in unpermeabilized specimens. In isoproterenol-treated cells, however, little receptor antigen was detected at the cell surface. Punctate receptor accumulations present in isoproterenol-treated cells were labeled by antibodies only following permeabilization of cells, as expected if these receptor accumulations were intracellular. Finally, internalized beta-adrenergic receptors colocalized with transferrin receptors, which are markers of endosomal membranes. These data provide several lines of evidence establishing that beta-adrenergic receptors undergo ligand-regulated internalization, they suggest that internalized receptors may be recycled back to the cell surface, and they provide the first direct indication that these processes involve the same endosomal membrane system passaged by constitutively recycling receptors.     

SRC regulates constitutive internalization and rapid resensitization of a cholecystokinin 2 receptor splice variant

The third intracellular loop domain of G protein-coupled receptors regulates their desensitization, internalization, and resensitization. Colorectal and pancreatic cancers, but not the nonmalignant tissue, express a splice variant of the cholecystokinin 2 receptor (CCK2R) called CCK(2i4sv)R that, because of intron 4 retention, contains an additional 69 amino acids within its third intracellular loop domain. This structural alteration is associated with agonist-independent activation of Src kinase (Olszewska-Pazdrak, B., Townsend, C. M., Jr., and Hellmich, M. R. (2004) J. Biol. Chem. 279, 40400-40404). The purpose of the study was to determine the roles of intron 4 retention and Src kinase on CCK(2i4sv)R desensitization, internalization, and resensitization. Gastrin1-17 (G17) binds to both CCK2R and CCK(2i4sv)R and induces intracellular Ca2+ ([Ca2+]i) increases. Agonist-induced increases in [Ca2+]i were used to assess receptor activity. Src kinase activity was inhibited by transducing cells with a retrovirus containing a dominant-negative mutant Src (A430V). The subcellular location of enhanced green fluorescent protein-tagged receptors was monitored using laser scanning confocal microscopy. Both receptor variants desensitized at the same rate; however, CCK(2i4sv)R resensitized five times faster than CCK2R. Without agonist, 80% of CCK(2i4sv)R is located in an intracellular compartment. In contrast, 80% of CCK2R was located on the plasma membrane. Treatment with inverse agonist (YM022) or expression of dominant-negative Src blocked the constitutive internalization of CCK(2i4sv)R, resulting in its accumulation on the plasma membrane. Expression of dominant-negative Src slowed the rate of CCK(2i4sv)R resensitization. Inhibition of Src did not affect G17-induced internalization of either receptor variant. Constitutive internalization of CCK(2i4sv)R increases its rate of resensitization by creating an intracellular pool of receptors that can rapidly recycle back to the plasma membrane.     

Transmembrane segment peptides can disrupt cholecystokinin receptor oligomerization without affecting receptor function

Oligomerization of the G protein-coupled cholecystokinin (CCK) receptor has been demonstrated, but its molecular basis and functional importance are not clear. We now examine contributions of transmembrane (TM) segments to oligomerization of this receptor using a peptide competitive inhibition strategy. Oligomerization of CCK receptors tagged at the carboxyl terminus with Renilla luciferase or yellow fluorescent protein was quantified using bioluminescence resonance energy transfer (BRET). Synthetic peptides representing TM I, II, V, VI, and VII of the CCK receptor were utilized as competitors. Of these, only TM VI and VII peptides disrupted receptor BRET. Control studies established that the beta2-adrenergic receptor TM VI peptide that disrupts oligomerization of that receptor had no effect on CCK receptor BRET. Notably, disruption of CCK receptor oligomerization had no effect on agonist binding, biological activity, or receptor internalization. To gain insight into the face of TM VI contributing to oligomerization, we utilized analogous peptides with alanines in positions 315, 319, and 323 (interhelical face) or 317, 321, and 325 (external lipid-exposed face). The Ala317,321,325 peptide eliminated the disruptive effect on CCK receptor BRET, whereas the other mutant peptide behaved like wild-type TM VI. This suggests that the lipid-exposed face of the CCK receptor TM VI most contributes to oligomerization and supports external contact dimerization of helical bundles, rather than domain-swapped dimerization. Fluorescent CCK receptor mutants with residues 317, 321, and 325 replaced with alanines were also prepared and failed to yield significant resonance transfer signals using either BRET or a morphological FRET assay, further supporting this interpretation.     

Comparative Localization of Receptors for Plasmin and for Urokinase on MCF7 Cells

The receptor for plasmin and the receptor for urokinase-type plasminogen activator were characterized on MCF 7 cells either independently or simultaneously, using fluorescence microscopy and confocal microscopy. The plasmin receptor was visualized, as previously described by Correc et al. (Int. J. Cancer 50, 767, 1992) using biotinylated plasminogen and fluoresceinated streptavidin. The urokinase receptor was revealed by both polyclonal and monoclonal antibodies reacting specifically with this receptor and by binding of urokinase aminoterminal fragment. On unfixed cells, these methods gave the same heterogeneous patterns of surface staining, consisting of contours and grains, localized mainly at the upper nonadherent face of the tumor cells by confocal microscopy. Only a part of the cells was stained. When both receptors were characterized together, their presence was found on the same cells and they gave almost superimposable patterns in many cases, as shown by confocal microscopy. In contrast, when MCF 7 cells were fixed or permeabilized before staining, quite different patterns were observed: almost all the cells were labeled. The staining was mainly cytoplasmic and localized preferentially in the center and close to the upper face of the cells. Similar results were found with antiserum against urokinase receptor and monoclonal antivinculin antibody. It is likely that the receptor for urokinase and the receptor for plasmin have similar localizations on MCF 7 cells, thus resulting in a functional cooperation.     

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.