In vivo and in vitro studies of cytosolic phospholipase A2 expression in Helicobacter pylori infection.

Modifications of mucosal phospholipids have been detected in samples from patients with Helicobacter pylori-positive gastritis. These alterations appear secondary to increased phospholipase A2 activity (PLA2). The cytosolic form of this enzyme (cPLA2), normally involved in cellular signaling and growth, has been implicated in cancer pathogenesis. The aim of this study was to investigate cPLA2 expression and PLA2 activity in the gastric mucosae of patients with and without H. pylori infection. In gastric biopsies from 10 H. pylori-positive patients, cPLA2 levels, levels of mRNA as determined by reverse transcriptase PCR, levels of protein as determined by immunohistochemistry, and total PLA2 activity were higher than in 10 H. pylori-negative gastritis patients. To clarify whether H. pylori had a direct effect on the cellular expression of cPLA2, we studied cPLA2 expression in vitro with different human epithelial cell lines, one from a patient with larynx carcinoma (i.e., HEp-2 cells) and two from patients with gastric adenocarcinoma (i.e., AGS and MKN 28 cells), incubated with different H. pylori strains. The levels of cPLA2, mRNA, and protein expression were unchanged in Hep-2 cells independently of cellular adhesion or invasion of the bacteria. Moreover, no change in cPLA2 protein expression was observed in AGS or MKN 28 cells treated with wild-type H. pylori. In conclusion, our study shows increased cPLA2 expression and PLA2 activity in the gastric mucosae of patients with H. pylori infection and no change in epithelial cell lines exposed to H. pylori.

Molecular characterization and organ distribution of type A and B cholecystokinin receptors in cynomolgus monkey.

Differences in the molecular structure or organ distribution of receptors can limit the usefulness of a given species for drug studies. In this work, we have studied cholecystokinin (CCK) receptors in cynomolgus monkey, an animal model useful for preclinical testing. The type A CCK receptor cDNA was cloned and predicted to encode a 428 amino acid peptide that was 98% identical to the human receptor. Only 2 of the 10 residues that were distinct from the human receptor were not present in other cloned CCK receptor species. A Chinese hamster ovary cell line that stably expressed this receptor was developed. The cynomolgus receptor expressed in this environment was functionally indistinguishable from the human receptor, binding CCK with high affinity [inhibition constant (K(I)) = 1.8 +/- 0.5 nM] and exhibiting a potent intracellular calcium signaling response to this hormone (EC(50) = 6.6 +/- 2.1 pM). Like the human type A CCK receptor, this receptor was expressed prominently in monkey gallbladder and stomach and was expressed in low levels in brain and pancreas. The type B CCK receptor cDNA was cloned from stomach and brain (450 residue receptor that is 96% identical to the human receptor), where it was highly expressed yet was undetectable in gallbladder or pancreas. This work confirms the relevance of the cynomolgus species for preclinical testing of drugs acting on the type A CCK receptor.

Refinement of the structure of the ligand-occupied cholecystokinin receptor using a photolabile amino-terminal probe.

Affinity labeling is a powerful tool to establish spatial approximations between photolabile residues within a ligand and its receptor. Here, we have utilized a cholecystokinin (CCK) analogue with a photolabile benzoylphenylalanine (Bpa) sited in position 24, adjacent to the pharmacophoric domain of this hormone (positions 27-33). This probe was a fully efficacious agonist that bound to the CCK receptor saturably and with high affinity (K(i) = 8.9 +/- 1.1 nm). It covalently labeled the CCK receptor either within the amino terminus (between Asn(10) and Lys(37)) or within the third extracellular loop (Glu(345)), as demonstrated by proteolytic peptide mapping, deglycosylation, micropurification, and Edman degradation sequencing. Truncation of the receptor to eliminate residues 1-30 had no detrimental effect on CCK binding, stimulated signaling, or affinity labeling through a residue within the pharmacophore (Bpa(29)) but resulted in elimination of the covalent attachment of the Bpa(24) probe to the receptor. Thus, the distal amino terminus of the CCK receptor resides above the docked ligand, compressing the portion of the peptide extending beyond its pharmacophore toward the receptor core. Exposure of wild type and truncated receptor constructs to extracellular trypsin damaged the truncated construct but not the wild type receptor, suggesting that this domain also may play a protective role. Use of these additional insights into molecular approximations provided key constraints for molecular modeling of the peptide-receptor complex, supporting the counterclockwise organization of the transmembrane helical domains.

Impaired resensitization and recycling of the cholecystokinin receptor by co-expression of its second intracellular loop.

Intermolecular interaction represents an important theme in regulation of intracellular trafficking of organelles that can be interrupted by competitive overexpression of a relevant molecular domain. We attempted to identify the functional importance of intracellular domains of the cholecystokinin (CCK) receptor by their over-expression in receptor-bearing Chinese hamster ovary (CHO-CCKR) cell lines. Although clathrin-dependent endocytosis and recycling of this receptor are well-established (J Cell Biol 128:1029-1042, 1995), any influence of distinct receptor domains is not understood. In this work, constructs representing each of the intracellular domains of the CCK receptor were coexpressed with wild-type receptor, and stable clonal cell lines were selected. Each was characterized for ligand binding and agonist-stimulated biological activity (inositol 1,4,5-trisphosphate generation), desensitization, resensitization, receptor internalization, and recycling. Each cell line expressed normal CCK radioligand binding, signaling, internalization, and desensitization. Three independent cell lines that coexpressed the 25-residue second intracellular loop domain exhibited deficient resensitization. In morphological assessment of receptor trafficking, this construct was also shown to interfere with receptor recycling to the plasma membrane. As a control, recycling of an unrelated G protein-coupled receptor was demonstrated to occur normally in this cell line. These observations suggest that rather than representing passive cargo within an endosome, a receptor can influence its own trafficking within the cell.

CCK receptor phosphorylation exposes regulatory domains affecting phosphorylation and receptor trafficking.

Agonist-stimulated phosphorylation of guanine nucleotide-binding protein (G protein)-coupled receptors has been recognized as an important mechanism for desensitization by interfering with coupling of the activated receptor with its G protein. We recently described a mutant of the CCK receptor that modified two of five key sites of phosphorylation (S260,264A) and eliminated agonist-stimulated receptor phosphorylation, despite normal ligand binding and signaling (20). As expected, this nonphosphorylated mutant had impaired rapid desensitization but was ultimately able to be desensitized by normal receptor internalization. Here we demonstrate that this mutant receptor is also defective in resensitization, with abnormal recycling to the cell surface. To explore this, another receptor mutant was prepared, replacing the same serines with aspartates to mimic the charge of serine-phosphate (S260,264D). This mutant was expressed in a Chinese hamster ovary cell line and shown to bind CCK normally. It had accelerated kinetics of signaling and desensitization and was phosphorylated in response to agonist occupation, with all other normal sites of phosphorylation modified. It was internalized like wild-type receptors and was resensitized and trafficked normally. This provides evidence for an additional important function for phosphorylation of G protein-coupled receptors. Phosphorylation may induce a conformational change in the receptor to expose other potential sites of phosphorylation and to expose domains involved in the targeting and trafficking of endosomes. The hierarchical phosphorylation of these sites may play a key role in receptor regulation.

Identification of a domain in the carboxy terminus of CCK receptor that affects its intracellular trafficking.

The carboxy-terminal region of many guanine nucleotide-binding protein (G protein)-coupled receptors contains important regulatory sequences such as an NP(x)2-3Y motif, a site of fatty acid acylation, and serine- and threonine-rich domains. The type A CCK receptor contains all of these, yet their significance has not been examined. We have, therefore, constructed a series of receptor site mutants and truncations that interfere with each of these motifs and expressed each in Chinese hamster ovary cells where they were studied for radioligand binding, cell signaling, receptor internalization, and intracellular trafficking. Each construct was synthesized and transported appropriately to the cell surface, where CCK bound with high affinity, elicited an inositol 1,4, 5-trisphosphate response, and resulted in internalization and normal trafficking. Thus modification or elimination of each of these established sequence motifs had no substantial effect on any of these parameters of receptor and cellular function. However, an additional construct that truncated the carboxy terminus, eliminating an additional 15-amino-acid segment devoid of any currently recognized sequence motifs, resulted in a marked change in receptor trafficking, with all other parameters of receptor function normal. This mutant receptor construct was delayed at the stage of early endosomes, delaying its progress to the lysosome-enriched perinuclear compartment from the rapid time course followed by wild-type receptor and all of the other constructs. It is proposed that this region of the CCK receptor tail contains a new motif important for intracellular receptor trafficking.

Direct identification of a second distinct site of contact between cholecystokinin and its receptor.

We have developed a biologically active analogue of cholecystokinin (CCK) that incorporates a photolabile benzoylphenylalanine (Bpa) moiety in the middle of its pharmacophoric domain, which efficiently establishes a covalent bond with an interacting domain of the CCK receptor. This probe incorporated L-Bpa in the position of Gly29 of the well characterized, radioiodinatable CCK analogue, D-Tyr-Gly-[(Nle28,31)CCK-26-33]. It was a potent pancreatic secretagogue (EC50 = 28 +/- 6 nM) that was equally efficacious with natural CCK, and bound to the CCK receptor with moderate affinity (IC50 = 450 +/- 126 nM). This was adequate to allow specific covalent labeling of the receptor. The labeled domain was within the cyanogen bromide fragment of the receptor including the top of TM6 (the sixth transmembrane domain), the third extracellular loop, and TM7 (the seventh transmembrane domain), as proven by direct Edman degradation sequencing. When this fragment was modified by the replacement of Val342 with Met to generate an additional site of cyanogen bromide cleavage, the labeled fragment was reduced in apparent size consistent with its representing the carboxyl-terminal portion of this fragment. Radiochemical sequencing of that fragment demonstrated covalent attachment of the probe to His347 and Leu348 in this domain. This represents the second experimentally demonstrated contact between a CCK analogue and this receptor, complementing the labeling of the domain just above TM1 (the first transmembrane domain) by a photolabile residue at the carboxyl terminus of CCK (Ji, Z. S., Hadac, E. M., Henne, R. M., Patel, S. A., Lybrand, T. P., and Miller, L. J. (1997) J. Biol. Chem. 272, 24393-24401). Both contacts are consistent with the conformational model of CCK binding proposed on the basis of the initial contact.

Differences in partial agonist action at cholecystokinin receptors of mouse and rat are dependent on parameters extrinsic to receptor structure: molecular cloning, expression and functional characterization of the mouse type A cholecystokinin receptor.

The mouse cholecystokinin (CCK) receptor is functionally distinct from the extensively studied rat receptor on the basis of differences in binding and biological activity of phenethyl ester analogs of CCK. These are partial agonists at the rat receptor and full agonists at the mouse pancreatic receptor. To explore this, we cloned the cDNA for the mouse type A CCK receptor, established a receptor-bearing Chinese hamster ovary (CHO) cell line and characterized its binding and biological characteristics. Despite 25 differences in amino acid sequence from the rat receptor, including a seven-amino acid insertion in the third intracellular loop, mouse and rat receptors were functionally indistinguishable when expressed in CHO cells. Of note, in the mouse pancreatic cell environment, a stable analog of guanosine triphosphate significantly inhibited binding of CCK-OPE, whereas it had no effect on binding to the same receptor on the CHO-CCKM cell line or to the rat receptor in either environment of the acinar cell. This likely reflects a difference in coupling of the mouse receptor to its G protein in the natural environment of the acinar cell. This may relate to differences extrinsic to the receptor, in the stoichiometry or character of G proteins or in the composition or organization of the lipid environment of the mouse acinar cell membrane. Although this may require complementation of the unique sequence of the mouse receptor, that structure alone is insufficient to explain this phenomenon. Receptor microenvironment makes an important, yet often ignored, contribution to receptor function.

Quantitative dynamic multicompartmental analysis of cholecystokinin receptor movement in a living cell using dual fluorophores and reconstruction of confocal images.

Receptor regulation is a key component of the phenomenon of desensitization in response to agonist stimulation which protects cells from overstimulation. Receptor internalization is one part of this response, often quantified by the portion of saturable ligand binding which becomes resistant to acidic washes. It is now clear that this can include receptor in multiple distinct cellular compartments. We have developed a morphological technique involving dual fluorescent probes to delineate the plasmalemma and the ligand-occupied receptor using confocal microscopy, with analysis involving three-dimensional reconstruction and quantitation of receptor movement through each compartment. When a radioiodinated cholecystokinin (CCK) analogue occupied its receptor on the CHO-CCKR cell line, it became progressively more resistant to dissociation with acidic medium. Quantitation of receptor internalization in these cells over time using this dynamic morphological technique correlated with the acid-resistant receptor fraction, and provided the additional information of the cellular compartments traversed. This technique will have multiple applications to explore the cell-specific handling of this and other ligand-occupied receptors.

Relationship between native and recombinant cholecystokinin receptors: role of differential glycosylation.

In an attempt to establish the relationship between the protein encoded by the recently cloned type A cholecystokinin (CCK) receptor cDNA and the two distinct plasmalemmal proteins on the rat pancreatic acinar cell that were previously described as candidates to represent this receptor, we have established a Chinese hamster ovary (CHO) cell line stably expressing large amounts of this recombinant protein and have used biochemical methods to characterize it directly. Upon affinity labeling, this protein migrated faster on a sodium dodecyl sulfate-polyacrylamide gel than the M(r) 85,000-95,000 molecule previously felt to represent the best candidate. However, deglycosylation with endoglycosidase F demonstrated that it had the same size core protein as that candidate, and this identification was further supported by protease peptide mapping. We postulated that the structural differences between the recombinant and the native proteins related to differences in glycosylation. Consistent with this, lectin-binding experiments demonstrated that both represented complex glycoproteins but that only the native receptor-bound Ulex europeus agglutinin I. Since this lectin binds to fucose residues that are added late in glycoprotein biosynthesis, it is possible that the distinct processing observed affected only that step. In spite of this structural difference, the type A CCK receptor-bearing CHO cell CCK receptor was functionally indistinguishable from the native acinar cell receptor. This included its ability to initiate signaling cascades, its sensitivity to stable GTP analogues, and its binding affinities for agonists and antagonists. The fidelity of this receptor expression system, while representing a 25-fold increase in receptor density over the native pancreatic acinar cell, should provide an ideal substrate for the examination of structure-function relationships within this molecule.

Abnormal processing of the human cholecystokinin receptor gene in association with gallstones and obesity.

BACKGROUND & AIMS: Cholesterol gallstone disease and obesity are often associated and share the potential, yet unreported, common etiology of cholecystokinin (CCK) dysfunction. While cloning the human CCK-A receptor complementary DNA (cDNA), we found predominance of a 262-base pair coding region deletion in a cDNA library prepared from a patient with this phenotype. The aim of this study was to determine the abundance, functional significance, and mechanism for generating this gene product. METHODS: Relative abundance of CCK receptor gene products was determined using polymerase chain reaction and hybridization analysis. Constructs were expressed in COS cells and studied for radioligand binding and intracellular calcium responses. A human genomic clone for this receptor was sequenced, and the critical regions were compared with those of the patient. RESULTS: Ninety-three percent of the patient's CCK receptor transcripts contained the 262-base pair deletion, whereas only 1.5% +/- 0.9% of control patients had the deletion. This encoded a receptor that did not bind or signal. The deletion corresponded with the third exon; however, this sequence and flanking introns were normal in the patient. CONCLUSIONS: Abnormality of processing an apparently normal CCK receptor gene yields the predominant product with an absent third exon and encoding a nonfunctional receptor, probably reflecting a defective trans-acting splicing factor. An atypical lariat region in the third intron may explain the presence of small amounts of this product in control patients.

Intrinsic photoaffinity labeling of native and recombinant rat pancreatic secretin receptors.

BACKGROUND: Structural characterization of pancreatic secretin receptors has been limited by difficulties in generating suitable radioligands and obtaining sufficient substrate. The aims of this study were to design, synthesize, and characterize high affinity radiolabeled analogues of secretin suitable for "intrinsic" photoaffinity labeling and to clone, express, and characterize the recombinant rat pancreatic secretin receptor. METHODS: The ability of synthetic analogues to stimulate amylase secretion by pancreatic acini was studied. Receptor complementary DNA (cDNA) was cloned by screening a rat pancreatic library with a probe based on the sequence of a neural cell secretin-binding protein. Competition binding and affinity labeling were performed with membranes prepared from rat pancreas and transfected cells. RESULTS: Two probes were fully efficacious secretagogues, which bound in a specific, high-affinity, rapid, and temperature-dependent manner. Only ([125I]Tyr10, pNO2-Phe22) rat secretin 27 covalently labeled a 50,000-62,000-molecular weight pancreatic membrane protein, with labeling inhibited in a concentration-dependent manner by secretin but not vasoactive intestinal polypeptide. Hybridization screening yielded a full-length cDNA identical to the neural clone. Photoaffinity labeling of this recombinant receptor identified a 57,000-62,000-molecular weight protein with specificity similar to that of native pancreas. Both native and recombinant receptors migrated at a molecular weight of 42,000 after endoglycosidase F deglycosylation. CONCLUSIONS: This study provides evidence for the molecular identity of the pancreatic secretin receptor and presents a novel probe important in structural characterization of its agonist-binding domain.

Conservation of coding and transcriptional control sequences within the snRNP E protein gene.

The snRNP E protein is one of several proteins associated with the U family of small nuclear RNAs that are involved in RNA processing. Isolation and characterization of the snRNP E protein cDNA sequences from mouse and chicken revealed a 100% conservation of the predicted amino acid sequence when compared to that of the human homologue. Further characterization of a genomic clone for the mouse snRNP E protein gene revealed that the 5' untranslated region and the immediate 5' upstream region have also been highly conserved: 72 and 70%, respectively. Conserved 5' regions include multiple copies of the CTTCCG hexamer sequence which are involved in regulating transcription of the human snRNP E protein gene. Mobility shift assays using corresponding DNA fragments from both human and mouse reveal that both fragments can compete for binding of at least one common transcription factor. These studies demonstrate that along with the amino acid sequence conservation between human and mouse, the snRNP E protein gene has also maintained a high DNA sequence conservation within its basal promoter structure.

A U6 snRNA gene with an internal promoter is juxtaposed to an snRNP protein sequence within an intron of a human G protein gene.

A complex locus on human chromosome 1 brings together sequences homologous to a G protein and two components of the RNA processing machinery of eukaryotic cells. Specifically, the seventh intron of the human Gi3 alpha gene contains a fusion of a partial snRNP E protein pseudogene to a variant U6 snRNA gene. The novel U6 sequence contains nine point mutations and a one nucleotide deletion relative to the major U6 genes from humans. Unlike all other vertebrate U6 genes characterized to date, the variant U6 gene is efficiently transcribed by RNA polymerase III even in the absence of all natural flanking sequences. The union of elements from the signal transduction pathway and the RNA processing machinery suggests the possibility of functional interplay.

The snRNP E protein multigene family contains five pseudogenes with common mutations.

Sequence data from three previously-uncharacterized members of the snRNP E protein multigene family suggest that each is a non-transcribed processed pseudogene, even though one clone has the potential to code for a full-length protein with greater than 90% similarity to previously-characterized E protein cDNAs. Each of the newly-analyzed family members is without introns, contains a tract of polyadenylic acid residues, and is flanked by short direct repeats. In addition, the three sequences all contain point mutations that distinguish them from the E protein coding sequence. Seven point mutations are common to the three sequences described here and to two previously-described E protein pseudogenes. Although all of these mutations are transitions, only 5 of 7 could have been generated by deamination of methylated cytosines in inactive genes. Thus, the common mutations in the pseudogenes suggest an origin other than the expressed gene that we have described. Allelic variants for two of the pseudogenes were detected and repetitive elements are located near four of the five E protein pseudogenes that have been characterized.

The small nuclear ribonucleoprotein E protein gene contains four introns and has upstream similarities to genes for ribosomal proteins.

The human small nuclear ribonucleoprotein E protein is an 11,000-dalton basic protein which is an integral component of several small nuclear ribonucleoprotein complexes involved in RNA processing reactions. Sequence analysis of the E protein multigene family reveals that at least one gene for this component of the RNA splicing machinery is interrupted by four introns. The exons of this gene are identical to two cDNA clones isolated from independent tissue sources and span approximately 9 kilobase pairs. Primer extension data indicated the presence of two major transcription start sites. The upstream region of the small nuclear ribonucleoprotein E protein gene does not contain TATA or CCAAT sequences within 175 nucleotides of the transcription start sites. However, the proximal upstream region does contain several similarities to the promoter regions of both snRNA genes and vertebrate ribosomal protein genes.

The complete primary structure of the human snRNP E protein.

The snRNP E protein is one of four "core" proteins associated with the snRNAs of the U family (U1,U2,U4,U5, and U6). Screening of a human teratoma cDNA library with a partial cDNA for a human autoimmune antigen resulted in the isolation of a cDNA clone containing the entire coding region of this snRNP core protein. Comparison of the 5' end of this cDNA with the sequences of two processed pseudogenes and primer extension data suggest that the cDNA is nearly full length. The longest open reading frame in this clone codes for a basic 92 amino acid protein which is in perfect agreement with amino acid sequence data obtained from purified E protein. The predicted sequence of this protein reveals no extensive similarity to other snRNP proteins, but contains regions of similarity to a eukaryotic ribosomal protein.

Relationship between androgen receptor binding activity in human prostate cancer and clinical response to endocrine therapy.

The authors investigated the ability of androgen receptor binding in prostate cancer tissue to predict the response of prostate cancer patients to endocrine therapy. The clinical response of 37 previously untreated patients with various grades and stages of prostate cancer was correlated with androgen receptor binding and detailed histologic data obtained before treatment. All patients underwent cold-punch transurethral resection of the prostate and received endocrine therapy. The association between time to progression and cytosolic androgen binding was not significant. However, the associations of time to progression to nuclear binding and to total androgen binding were significant (P = 0.029 and 0.038, respectively). The authors found no association between clinical stage and time to progression, but did find an association between time to progression and pathologic grade (P = 0.003); grade 4 lesions were the least responsive to hormone therapy. When grade 4 lesions were excluded (N = 3), binding levels were still predictive of progression independently of grade and stage. The authors conclude that nuclear receptor binding activity in localized and metastatic prostate cancer tissue is predictive of response to hormonal manipulation.