Pharmacological characterization of cholecystokinin receptors mediating contraction of human gallbladder and ascending colon

Cholecystokinin (CCK) produces contractions of gallbladder and colon in a number of different species. Although the effects of CCK on the human gallbladder are relatively well documented, the CCK receptors in the human colon have not been clearly characterised. Therefore, in this study, the CCK receptors in the human gallbladder and colon were compared using pharmacological techniques. Contraction of specimens of the human tissue was measured using in vitro organ bath bioassay. The effect of selective concentrations of CCK(1) and CCK(2) receptor antagonists (L-364,718 and JB93182, respectively) was determined on agonist concentration-effect (E/[A]) curves obtained by cumulative dosing with sulphated CCK. The CCK(1) antagonist L-364,718 produced a rightward shift of the CCK-8S [E/[A] curve in the human gallbladder (pA(2)=9.15 +/- 0.26) and ascending colon (pA(2)=9.20 +/- .33). In both tissues, the CCK(2) receptor antagonist, JB93182, had no effect on the CCK E/[A] curves. In addition, in the colon, pentagastrin responses were inhibited by L-364,718 but unaffected by JB93182. These data indicate that the CCK-induced contraction of the human colon and gallbladder smooth muscle is mediated solely through the CCK(1) receptor subtype, and the antagonist affinity estimates are consistent with those previously obtained in experiments on animal tissue.     

Pharmacological analysis of CCK(2) receptor ligands using COS-7 and SK-N-MC cells, expressing the human CCK(2) receptor.

A series of CCK(2) receptor ligands were analysed with respect to their interaction with binding sites in the membranes of COS-7 cells and SK-N-MC cells transiently expressing the human CCK(2) receptor (short isoform). The ligands were YF476, YM022, AG041R, L-740,093, JB93182, PD134308, and PD136450. Their binding was analysed by radioligand competition using [3H]L-365,260 as the labelled ligand. Saturation binding analysis indicated that [3H]L-365,260 interacted with a single class of binding sites. In competition binding experiments using COS-7-cell membranes, all seven ligands were incubated together with 2 nM [3H]L-365,260. The data for four of the compounds fitted a one-site model (pK(i) values: YM022: 9.2+/-0.02; YF476: 9.6+/-0.04; L-740,093: 9.2+/-0.01; and AG041R: 8.3+/-0.06), while the data for the three others fitted a two-site model (pK(i) values: JB93182: 8.8+/-0.04 and 6.0+/-0.15; PD134308: 9.0+/-0.04 and 6.1+/-0.15; and PD136450: 9.0+/-0.02 and 5.4+/-0.41). SK-N-MC cell membranes and 2 nM [3H]L-365,260 were incubated together with YM022, YF476, JB93182, and PD134308. The data for YM022 and YF476 fitted a one-site model (pK(i) values: YM022: 9.3+/-0.06; YF476: 9.4+/-0.02), while the data for JB93182 and PD134308 fitted a two-site model (pK(i) values: JB93182: 8.7+/-0.06 and 6.2+/-0.06; PD134308: 9.1+/-0.06 and 7.0+/-0.17). Competition binding experiments in the presence of the GTP-analogue guanylylimidodiphosphate, using either of the two cell types, produced similar binding data for PD134308 and JB93182 as in the absence of GTP-analogue. The human receptor seems to exist in a low and/or high affinity state. The shift from low to high affinity does not seem to reflect the degree of G protein coupling.     

Effects of DNA binding of the zinc finger and linkers for domain fusion on the catalytic activity of sequence-specific chimeric recombinases determined by a facile fluorescent system

Artificial zinc finger proteins (ZFPs) consist of Cys(2)-His(2)-type modules composed of ～30 amino acids with a ββα structure that coordinates a zinc ion. ZFPs that recognize specific DNA target sequences can substitute for the binding domains of enzymes that act on DNA to create designer enzymes with programmable sequence specificity. The most studied of these engineered enzymes are zinc finger nucleases (ZFNs). ZFNs have been widely used to model organisms and are currently in human clinical trials with an aim of therapeutic gene editing. Difficulties with ZFNs arise from unpredictable mutations caused by nonhomologous end joining and off-target DNA cleavage and mutagenesis. A more recent strategy that aims to address the shortcomings of ZFNs involves zinc finger recombinases (ZFRs). A thorough understanding of ZFRs and methods for their modification promises powerful new tools for gene manipulation in model organisms as well as in gene therapy. In an effort to design efficient and specific ZFRs, the effects of the DNA binding affinity of the zinc finger domains and the linker sequence between ZFPs and recombinase catalytic domains have been assessed. A plasmid system containing ZFR target sites was constructed for evaluation of catalytic activities of ZFRs with variable linker lengths and numbers of zinc finger modules. Recombination efficiencies were evaluated by restriction enzyme analysis of isolated plasmids after reaction in Escherichia coli and changes in EGFP fluorescence in mammalian cells. The results provide information relevant to the design of ZFRs that will be useful for sequence-specific genome modification.     

Arginine 197 of the cholecystokinin-A receptor binding site interacts with the sulfate of the peptide agonist cholecystokinin

The knowledge of the binding sites of G protein-coupled cholecystokinin receptors represents important insights that may serve to understand their activation processes and to design or optimize ligands. Our aim was to identify the amino acid of the cholecystokinin-A receptor (CCK-AR) binding site in an interaction with the sulfate of CCK, which is crucial for CCK binding and activity. A three-dimensional model of the [CCK-AR-CCK] complex was built. In this model, Arg197 was the best candidate residue for a ionic interaction with the sulfate of CCK. Arg197 was exchanged for a methionine by site-directed mutagenesis. Wild-type and mutated CCK-AR were transiently expressed in COS-7 cells for pharmacological and functional analysis. The mutated receptor on Arg197 did not bind the agonist radioligand 125I-BH-[Thr, Nle]-CCK-9; however, it bound the nonpeptide antagonist [3H]-SR27,897 as the wild-type receptor. The mutant was approximately 1,470- and 3,200-fold less potent than the wild-type CCK-AR to activate G proteins and to induce inositol phosphate production, respectively. This is consistent with the 500-fold lower potency and 800-fold lower affinity of nonsulfated CCK relative to sulfated CCK on the wild-type receptor. These data, together with those showing that the mutated receptor failed to discriminate nonsulfated and sulfated CCK while it retained other pharmacological features of the CCK-AR, strongly support an interaction between Arg197 of the CCK-AR binding site and the sulfate of CCK. In addition, the mutated CCK-AR resembled the low affinity state of the wild-type CCK-AR, suggesting that Arg197-sulfate interaction regulates conformational changes of the CCK-AR that are required for its physiological activation.     

Selectivity of arsenite interaction with zinc finger proteins

Arsenic is a carcinogenic element also used for the treatment of acute promyelocytic leukemia. The reactivity of proteins to arsenic must be associated with the various biological functions of As. Here, we investigated the selectivity of arsenite to zinc finger proteins (ZFPs) with different zinc binding motifs (C2H2, C3H, and C4). Single ZFP domain proteins were used for the direct comparison of the reactivity of different ZFPs. The binding constants and the reaction rates have been studied quantitatively. Results show that both the binding affinity and reaction rates of single-domain ZFPs follow the trend of C4 > C3H ? C2H2. Compared with the C2H2 motif ZFPs, the binding affinities of C3H and C4 motif ZFPs are nearly two orders of magnitude higher and the reaction rates are approximately two-fold higher. The formation of multi-domain ZFPs significantly enhances the reactivity of C2H2 type ZFPs, but has negligible effects on C3H and C4 ZFPs. Consequently, the reactivities of the three types of multi-domain ZFPs are rather similar. The 2D NMR spectra indicate that the As(III)-bound ZFPs are also unfolded, suggesting that arsenic binding interferes with the function of ZFPs.     

Characterization of [3H]naltrindole binding to delta opioid receptors in rat brain.

[3H]Naltrindole binding characteristics were determined using homogenized rat brain tissue. Saturation binding studies at 25 degrees C measured an equilibrium dissociation constant (Kd) value of 37.0 +/- 3.0 pM and a receptor density (Bmax) value of 63.4 +/- 2.0 fmol/mg protein. Association binding studies showed that equilibrium was reached within 90 min at a radioligand concentration of 30 pM. Naltrindole, as well as the ligands selective for delta (delta) opioid receptors, such as pCI-DPDPE and Deltorphin II inhibited [3H]naltrindole binding with nanomolar IC50 values. Ligands selective for mu (mu) and kappa (kappa) opioid receptors were only effective in inhibiting [3H]naltrindole binding at micromolar concentrations. From these data, we conclude that [3H]naltrindole is a high affinity, selective radioligand for delta opioid receptors.     

The utilization of recombinant prostanoid receptors to determine the affinities and selectivities of prostaglandins and related analogs

Stable cell lines that individually express the eight known human prostanoid receptors (EP(1), EP(2), EP(3), EP(4), DP, FP, IP and TP) have been established using human embryonic kidney (HEK) 293(EBNA) cells. These recombinant cell lines have been employed in radioligand binding assays to determine the equilibrium inhibitor constants of known prostanoid receptor ligands at these eight receptors. This has allowed, for the first time, an assessment of the affinity and selectivity of several novel compounds at the individual human prostanoid receptors. This information should facilitate interpretation of pharmacological studies that employ these ligands as tools to study human tissues and cell lines and should, therefore, result in a greater understanding of prostanoid receptor biology.     

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.     

Somatostatin receptors on rat pancreatic acinar cells. Pharmacological and structural characterization and demonstration of down-regulation in streptozotocin diabetes

The binding of somatostatin-14 (S-14) to rat pancreatic acinar cell membranes was characterized using [125I-Tyr11]S-14 as the radioligand. Maximum binding was observed at pH 7.4 and was Ca2+-dependent. Such Ca2+ dependence of S-14 receptor binding was not observed in other tissues. Scatchard analysis of the competitive inhibition by S-14 of [125I-Tyr11]S-14 binding revealed a single class of high affinity sites (Kd = 0.5 +/- 0.07 nM) with a binding capacity (Bmax) of 266 +/- 22 fmol/mg of protein. [D-Trp8]S-14 and structural analogs with halogenated Trp moiety exhibited 2-32-fold greater binding affinity than S-14, [D-F5-Trp8]S-14 being the most potent. [Tyr11]S-14 was equipotent with S-14. The affinity of somatostatin-28 for binding to these receptors was 50% of that of S-14. Cholecystokinin octapeptide (CCK-8) inhibited the binding of [125I-Tyr11]S-14, but its inhibition curve was not parallel to that of S-14. In the presence of 1 nM CCK-8, the Bmax of S-14 receptors was reduced to 150 +/- 17 fmol/mg of protein. Dibutyryl cyclic GMP, a CCK receptor antagonist, partially reversed the inhibitory action of CCK-8, suggesting that CCK receptors mediate the inhibition of S-14 receptor binding. GDP, GTP, and guanyl-5'-yl imidodiphosphate inhibit S-14 receptor binding in this tissue. The inhibition was shown to be due to decrease in binding capacity and not due to change in affinity. Specifically bound [125I-Tyr11]S-14 cross-linked to the S-14 receptors was found associated with three proteins of approximate Mr = 200,000, 80,000, and 70,000 which could be detected under both reducing and nonreducing conditions. Finally, pancreatic acinar cell S-14 receptors were shown to be down-regulated by persistent hypersomatostatinemia 1 week after streptozotocin-induced diabetes characterized by decreased Bmax (105 +/- 13 fmol/mg of protein) without any change in affinity. We conclude that pancreatic acinar cell membrane S-14 receptors require Ca2+ for maximal binding and thus differ from S-14 receptors in other tissues, S-14 receptors in this tissue also exhibit selective ligand specificities, these receptors are regulated by CCK-8 and guanine nucleotides, three receptor proteins of apparent Mr = 200,000, 80,000, and 70,000 specifically bind S-14, and (v) these receptors are regulated by S-14 in vivo as evidenced by decreased binding in streptozotocin diabetic rats characterized by hypersomatostatinemia.     

Towards therapeutic applications of engineered zinc finger proteins

It has long been the goal of molecular biologists to design DNA-binding proteins for the specific control of gene expression. The zinc finger design is ideally suited for such purposes, discriminating between closely related sequences both in vitro and in vivo. Whereas other DNA-binding proteins generally make use of the 2-fold symmetry of the double helix, zinc fingers do not and so can be linked linearly in tandem to recognize DNA sequences of different lengths, with high fidelity. This modular design offers a large number of combinatorial possibilities for the specific recognition of DNA. By fusing zinc finger peptides to repression or activation domains, genes can be selectively targeted and switched off and on. Several recent applications of such engineered zinc finger proteins (ZFPs) are described, including the activation of vascular endothelial growth factor (VEGF) in a human cell line and an animal model. Clinical trials have recently begun on using VEGF-activating ZFPs to treat human peripheral arterial disease, by stimulating vascular growth. Also in progress are pre-clinical studies using ZFPs to target the defective genes in two monogenic disorders, SCID and SCA. The aim is to replace them in each case by a correct copy from an extrachromosomal DNA donor by means of homologous recombination. Promising results are reported.     

Linking non-peptide ligand binding mode to activity at the human cholecystokinin-2 receptor

Given the importance of G-protein-coupled receptors as pharmacological targets in medicine, efforts directed at the understanding the molecular mechanism by which pharmacological compounds regulate their activity is of paramount importance. Here, we investigated at an atomic level the mechanism of inverse agonism and partial agonism of two high affinity, high selectivity very similar non-peptide ligands of the cholecystokinin-2 receptor (CCK2R) which differ by the absence or presence of a methyl group on their indole moiety. Using in silico, site-directed mutagenesis and pharmacological experiments, we demonstrated that these functionally different activities are due to differing anchoring modes of the two compounds to a residue of helix II (Thr-2.61) in the inactive state of the CCK2R. The binding mode of the inverse agonist allows the ligand to interact through its phenyl moiety with a key amino acid for CCK2R activation (Trp-6.48), preventing rotation of helix VI and, thus, CCK2R activation, whereas the partial agonist binds deeper into the binding pocket and closer to helix V, so that CCK2R activation is favored. This study on the molecular mechanism of ligand action opens the possibility of target-based optimization of G protein-coupled receptor non-peptide ligands.     

Receptors for cholecystokinin and gastrin peptides display specific binding properties and are structurally different in guinea-pig and dog pancreas

In the light of the strong potency of gastrin-related peptides on pancreatic exocrine secretion in dog, we analyzed the binding properties of peptides related to cholecystokinin (CCK) and gastrin on dog pancreatic acini compared to guinea-pig acini. Moreover, we determined apparent molecular masses of photoaffinity labelled CCK/gastrin receptors in the two models. Using the CCK radioligand, receptor selectivity towards CCK/gastrin agonists and antagonists was found to be lower in dog acini than in guinea-pig acini. Performing the binding with CCK and gastrin radioligands in combination with N2,O2'-dibutyryl-guanosine 3',5'-monophosphate, revealed that in dog acini there exist two different sub-classes of CCK/gastrin receptors having high and low selectivity, the latter ones being able to bind gastrin with high affinity (Kd = 2.1 nM). SDS-PAGE analysis of covalently cross-linked receptors using several photosensitive CCK and gastrin probes of different peptide chain lengths demonstrated that in guinea-pig, CCK peptides bound to a 84-kDa component whereas in dog pancreas, CCK and gastrin peptides bound to three distinct molecular species (Mr approximately equal to 78,000, 45,000, 28,000). Performing cross-linking in the presence of 1 microM CCK indicated that a 45-kDa protein is the putative CCK/gastrin receptor in dog pancreas. Our results support the concept of heterogeneity of CCK/gastrin receptors.     

Identification and characterization of leukotriene D4 receptors and signal transduction processes in rat basophilic leukemia cells

The leukotriene D4 (LTD4) receptor on rat basophilic leukemia (RBL-1) cell membranes was characterized using a radioligand binding assay. [3H]LTD4 binding to RBL-1 membrane receptors was stereoselective, specific, and saturable. The binding affinity and maximum binding density of [3H]LTD4 to RBL-1 membrane receptors were 0.9 +/- 0.2 nM and 800 +/- 125 fmol/mg protein, respectively. Binding of [3H]LTD4 to the receptors was enhanced by divalent cations (Ca2+, Mg2+, and Mn2+) and inhibited by guanine nucleotides and sodium ions, specifically, indicating that a guanine nucleotide-binding protein may regulate the agonist-receptor interaction. LTD4, LTE4 agonist and antagonist analogs competed with the radioligand in binding to the RBL-1 LTD4 receptors. The binding affinities of these analogs correlated with (a) those determined from the guinea pig lung LTD4 receptors and (b) the pharmacological activities in smooth muscle contraction. LTD4 and related agonists also induced time- and concentration-dependent phosphatidylinositol hydrolysis in RBL-1 cells. The LTD4 induction of inositol 1-phosphate was potent, stereoselective, specific, and was blocked by LTD4 receptor antagonists. The rank order potency of agonist-induced inositol 1-phosphate formation in RBL-1 cells was equivalent to the receptor binding affinity determined using either RBL-1 cell or guinea pig lung membranes. These studies have demonstrated the G protein coupled LTD4 receptors on RBL-1 cell membranes. Binding of agonists to the receptor may activate the G protein-regulated phospholipase C to induce hydrolysis of phosphatidylinositol. The hydrolytic products of phosphatidylinositol, possibly inositol trisphosphate and diacylglycerol, may be the intracellular messengers for LTD4 receptors in RBL-1 cells.     

Dopamine binding to the alpha receptor in pregnant rabbit myometrium

This study evaluated in vitro binding of dopamine ligands to myometrial alpha adrenoceptors. With cell membranes from pregnant rabbits, receptor radioligand binding studies utilizing [3H] dihydroergocryptine +/- dopamine demonstrated receptor affinity (KD) = 0.75 +/- 0.10 nM (+/- SEM) and density (Bmax) = 533.2 +/- 45.2 fM/mg protein. Similar studies utilizing phentolamine or apomorphine gave essentially identical results. Competition binding studies demonstrated steriospecific butaclamol binding, along with significant binding of haloperidol, spiperone, apomorphine, and bromoergocryptine. These observations provide a mechanism for the observed uterotonic effects of dopamine.     

In vitro characterization of [3H]MethoxyPyEP,an mGluR5 selective radioligand

We have characterized the in vitro properties of 3-[3H]methoxy-5-(pyridin-2-ylethynyl)pyridine ([3H]MethoxyPyEP), an analogue of the mGluR(5) receptor subtype antagonist MPEP [2-methyl-6-(phenylethynyl)-pyridine], in rat tissue preparations using tissue homogenates and autoradiography. Binding of [3H]MethoxyPyEP to rat cortex, hippocampus, thalamus and cerebellum membrane preparations revealed saturable, high affinity binding (3.4 +/- 0.4 nM, n = 4 in rat cortex) to a single population of receptors in all regions studied except for cerebellum. Binding was found to be relatively insensitive to pH and insensitive to DTT. High concentrations of NEM both reduce receptor concentration and binding affinity for the radioligand. In time-course studies at room temperature k(on) and k(off) were determined as 2.9 x 10(7) M(-1) min(-1) and 0.11 min(-1) respectively. The rank order of affinities, as assessed by equilibrium competition studies, of a variety of ligands suggested binding of the radioligand selectively to mGluR5 (MPEP > trans-azetidine-2,4-dicarboxylic acid congruent with (S)-4-carboxyphenylglycine congruent with (+)MK801 congruent with CP-101,606 congruent with clozapine congruent with atropine congruent with ketanserin congruent with yohimbine congruent with benoxathian). Autoradiographic studies with [3H]MethoxyPyEP showed that binding was regioselective, with high density of binding in caudate and hippocampus, intermediate binding in thalamus and very low density in the cerebellum. These data show that [3H]MethoxyPyEP is a high affinity radioligand useful for the in vitro study of mGluR5 receptor distribution and pharmacologic properties in brain.     

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.     

Binding of a phenethyl ester analogue of cholecystokinin to the solubilized pancreatic cholecystokinin receptor: use in ligand-affinity chromatography.

Pancreatic acinar cells have both high and low affinity receptors for cholecystokinin (CCK), yet their membranes appear to possess only a single class of binding sites. Recently, gallbladder membrane CCK receptors were shown to undergo inter-conversion between two affinity states dependent on G protein coupling. Keys for that observation were the differential binding affinities of CCK and a phenethyl ester analogue of CCK (OPE), with the high affinity state binding CCK with higher affinity than OPE, and the low affinity state binding OPE with higher affinity than CCK. Here, we performed analogous experiments using these ligands and both pancreatic membranes and a solubilized preparation. Both preparations were found to have only single affinity states of this receptor. However, the state on membranes had a higher affinity for CCK than for OPE, and that on the solubilized preparation had a higher affinity for OPE than for CCK. This supports the hypothesis that the ternary complex of ligand-receptor-G protein found in membranes represents the high affinity state of this receptor, while the uncoupled form of this receptor after solubilization represents its low affinity state. The high affinity of OPE for the solubilized receptor can be utilized in a purification strategy to follow receptor-bearing fractions and to provide an efficient and specific affinity-binding step.