Studies of relationships between variation of the human G protein-coupled receptor 40 Gene and Type 2 diabetes and insulin release.

AIMS: Recently, a novel human G protein-coupled receptor 40 (GPR40), which is predominantly expressed in pancreatic islets, was shown to mediate an amplifying effect of long-chain fatty acids on glucose-induced insulin secretion. The present aim was to examine the coding region of GPR40 for variation and to assess whether identified variants confer an increased risk of Type 2 diabetes or altered insulin release. METHODS: Mutation analysis was performed in 43 patients with Type 2 diabetes, 18 normal glucose-tolerant subjects, and 3 maturity-onset of diabetes in the young (MODY) X patients using direct sequencing. Genotyping was performed using polymerase chain reaction (PCR)-generated primer extension products analysis by high throughput chip-based mass spectrometry (MALDI-TOF). The potential impact of GPR40 mutations on [(3)H]-myo-inositol turnover was estimated in COS-7 cells after stimulation with various concentrations of 5,8,11-eicosatriynoic acid. RESULTS: Two nucleotide substitutions, an Arg211His polymorphism and a rare Asp175Asn mutation, were identified. Both variants showed EC(50) values similar to the wild type. However, the maximal efficacy of the rare Asp175Asn was 39% lower compared with the wild type (P = 0.01). The Arg211His polymorphism had a similar allele frequency among 1384 Type 2 diabetic patients [MAF%; 23.4 (95% CI: 21.8-25.0)] and 4424 middle-aged glucose-tolerant subjects [24.1% (23.2-25.0)]. A genotype-quantitative trait study of 5597 non-diabetic, middle-aged subjects from the Inter99 cohort showed no significant differences in oral glucose tolerance test (OGTT)-derived estimates of insulin release between carriers of various GPR40 genotypes. CONCLUSIONS: Variations in the coding region of GPR40 do not appear to be associated with Type 2 diabetes or insulin release alterations.

Characterization of the human Fc gamma RIIB gene promoter: human zinc-finger proteins (ZNF140 and ZNF91) that bind to different regions function as transcription repressors.

Expression of the human low-affinity Fc receptors for IgG (human Fc gamma RII) is differentially regulated. We report here the characterization of the promoter structure of the human Fc gamma RIIB gene and the isolation of the promoter region-binding proteins by a yeast one-hybrid assay. The minimal 154-bp region upstream from the transcription start site of the human Fc gamma RIIB gene was shown to possess promoter activity in a variety of cells. An electrophoretic mobility shift assay indicated that multiple nuclear factors in cell extracts bind to the two regions [F2-3 (-110 to -93) and F4-3 (-47 to -31)] of the human Fc gamma RIIB gene promoter. Mutation analysis indicated that GGGAGGAGC (-105 to -97) and AATTTGTTTGCC (-47 to -36) sequences are responsible for binding to nuclear factors respectively. By using GGGAGGAGC and AATTTGTTTGCC as bait sequences, we cloned two zinc-finger proteins (ZNF140 and ZNF91) that bind to the F2-3 and F4-3 regions within the promoter of the human Fc gamma RIIB gene respectively. When the ZNF140 and ZNF91 were transfected with reporter plasmid, both showed repressor activity with additive effects. Thus, these results indicate that these cloned ZNF140 and ZNF91 proteins function as repressors for the human Fc gamma RIIB transcription.

Structure of Fab hGR-2 F6, a competitive antagonist of the glucagon receptor.

The monoclonal antibody hGR-2 F6 has been raised against the human glucagon receptor and shown to act as a competitive antagonist. As a first step in the structural characterization of the receptor, the crystal structure of the Fab fragment from this antibody is reported at 2.1 A resolution. The hGR-2 F6 Fab crystallizes in the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 76.14, b = 133.74, c = 37.46 A. A model generated by homology modelling was used as an aid in the chain-tracing and the Fab fragment structure was subsequently refined (final R factor = 21.7%). The structure obtained exhibits the typical immunoglobulin fold. Complementarity-determining regions (CDRs) L1, L2, L3, H1 and H2 could be superposed onto standard canonical CDR loops. The H3 loop could be classified according to recently published rules regarding loop length, sequence and conformation. This loop is 14 residues long, with an approximate beta-hairpin geometry, which is distorted somewhat by the presence of two trans proline residues at the beginning of the loop. It is expected that this H3 loop will facilitate the design of synthetic probes for the glucagon receptor that may be used to investigate receptor activity.

Human ERRgamma, a third member of the estrogen receptor-related receptor (ERR) subfamily of orphan nuclear receptors: tissue-specific isoforms are expressed during development and in the adult.

The nuclear receptor protein superfamily is a large group of transcription factors involved in many aspects of animal development, tissue differentiation, and homeostasis in the higher eukaryotes. A subfamily of receptors, ERRalpha and beta (estrogen receptor-related receptor alpha and beta), closely related to the ER, were among the first orphan nuclear receptors identified. These receptors can bind DNA as monomers and are thought to activate transcription constitutively, unaffected by beta-estradiol. Studies of the expression patterns of ERRalpha and gene disruption experiments of ERRbeta indicate that they play an important role in the development and differentiation of specific tissues in the mouse. In this work we demonstrate the existence in humans of a third member of this subfamily of receptors, termed ERRgamma, which is highly expressed in a number of diverse fetal and adult tissues including brain, kidney, pancreas, and placenta. The ERRgamma mRNA is highly alternatively spliced at the 5'-end, giving rise to a number of tissue-specific RNA species, some of which code for protein isoforms differing in the N-terminal region. Like ERRalpha and beta, ERRgamma binds as a monomer to an ERRE. A GAL4-ERRgamma fusion protein activates transcription in a ligand-independent manner in transfected HEK293 cells to a greater degree than either the GAL4-ERRalpha or -beta fusion proteins.

Mining of assembled expressed sequence tag (EST) data for protein families: application to the G protein-coupled receptor superfamily.

The availability of large expressed sequence tag (EST) databases has led to a revolution in the way new genes are identified. Mining of these databases using known protein sequences as queries is a powerful technique for discovering orthologous and paralogous genes. The scientist is often confronted, however, by an enormous amount of search output owing to the inherent redundancy of EST data. In addition, high search sensitivity often cannot be achieved using only a single member of a protein superfamily as a query. In this paper a technique for addressing both of these issues is described. Assembled EST databases are queried with every member of a protein superfamily, the results are integrated and false positives are pruned from the set. The result is a set of assemblies enriched in members of the protein superfamily under consideration. The technique is applied to the G protein-coupled receptor (GPCR) superfamily in the construction of a GPCR Resource. A novel full-length human GPCR identified from the GPCR Resource is presented, illustrating the utility of the method.

Retinoic acid down-regulates the expression of the vasoactive intestinal polypeptide receptor type-1 in human breast carcinoma cell lines.

Four breast carcinoma cell lines (T47D, ZR-75-1, MDA-MB-231, and MCF-7) were tested for regulation of the expression of vasoactive intestinal polypeptide receptor type-1 (VIP-R1). In all four cell lines, retinoic acid (RA) treatment caused a fast and marked decrease in VIP-R1 mRNA level as examined by Northern blots. Cycloheximide pretreatment attenuated the effect from 3- to 2-fold, indicating that existing proteins can mediate the decreasing effect of RA, but to attain the maximal effect new protein synthesis might be needed. Transcriptional inhibition with Actinomyocin D showed that RA did not influence the VIP-R1 mRNA half-life, indicating that the decreasing effect of RA on the mRNA level is due to transcriptional inhibition. In agreement with the observations on mRNA level, we found that the VIP receptor number was reduced 3-fold from 88 to 32 fmol/10(6) cells in T47D cells and from 222 to 73 fmol/10(6) cells in MDA-MB-231 cells upon RA treatment for 72 h. The promoter and 5'-flanking region of the VIP-R1 gene were cloned from a human placental cosmid library, and 2.5 kb were sequenced to search for regulatory elements. Our results, therefore, imply that the regulation of VIP-R1 gene expression by RA could have a role in human mammary tumor biology.

Ciao 1 is a novel WD40 protein that interacts with the tumor suppressor protein WT1.

The Wilms tumor suppressor protein, WT1, is a transcription factor capable of activating or repressing transcription of various cellular genes. The mechanisms involved in regulating the transcriptional activities of WT1 are beginning to be unraveled. It appears that physical interactions of other cellular proteins (p53 and par-4) with WT1 can modulate the function of WT1. Here, we report the identification and cloning of a novel WT1-interacting protein termed Ciao 1, a member of the WD40 family of proteins. Ciao 1 specifically interacts with WT1 both in vitro and in vivo. This interaction alters the mobility of a WT1.DNA complex in gel shift assays, and results in a decrease in transcriptional activation mediated by WT1. Ciao 1 does not inhibit binding of WT1 to its consensus nucleotide sequence and does not affect the repression activity of WT1. Thus, Ciao 1 appears to specifically modulate the transactivation activity of WT1 and may function to regulate the physiological functions of WT1 in cell growth and differentiation.

BAP1: a novel ubiquitin hydrolase which binds to the BRCA1 RING finger and enhances BRCA1-mediated cell growth suppression.

We have identified a novel protein, BAP1, which binds to the RING finger domain of the Breast/Ovarian Cancer Susceptibility Gene product, BRCA1. BAP1 is a nuclear-localized, ubiquitin carboxy-terminal hydrolase, suggesting that deubiquitinating enzymes may play a role in BRCA1 function. BAP1 binds to the wild-type BRCA1-RING finger, but not to germline mutants of the BRCA1-RING finger found in breast cancer kindreds. BAP1 and BRCA1 are temporally and spatially co-expressed during murine breast development and remodeling, and show overlapping patterns of subnuclear distribution. BAP1 resides on human chromosome 3p21.3; intragenic homozygous rearrangements and deletions of BAP1 have been found in lung carcinoma cell lines. BAP1 enhances BRCA1-mediated inhibition of breast cancer cell growth and is the first nuclear-localized ubiquitin carboxy-terminal hydrolase to be identified. BAP1 may be a new tumor suppressor gene which functions in the BRCA1 growth control pathway.

Assignment of human elongation factor 1alpha genes: EEF1A maps to chromosome 6q14 and EEF1A2 to 20q13.3.

The human elongation factor 1alpha gene family consists of at least 2 actively transcribed genes, EEF1A and EEF1A2, and more than 18 homologous loci. EEF1A2 is expressed in a tissue-specific manner, whereas EEF1A is expressed ubiquitously, and both of them can function in translation. An EEF1A cDNA probe has previously been shown to cross-hybridize with several human chromosomes, but the location of the functional gene has not been established. We have mapped the functional EEF1A gene to 6q14 by combined fluorescence in situ hybridization (FISH) and PCR analysis of a somatic cell hybrid panel and mapped EEF1A2 to 20q13.3 by FISH. In addition, the 11 strongest cross-hybridizing loci (EEF1AL2-EEF1AL13) were mapped by FISH to 12p12, 9q34, 7p15-p21, 19q13, 3q26-q27, 7q33-q35, 1p13-p22, 2q12-q14, 5p12-q11, 1q31-q32, and Xq21.

Hyperproinsulinemia in a three-generation Caucasian family due to mutant proinsulin (Arg65-His) not associated with imparied glucose tolerance: the contribution of mutant proinsulin to insulin bioactivity.

Familial hyperproinsulinemia is a genetic abnormality characterized by an increased proportion of proinsulin immunoreactivity in the circulation due to mutations affecting the posttranslational processing of proinsulin. In affected Japanese families, this has been associated with noninsulin-dependent diabetes mellitus or impaired glucose tolerance. A three-generation Caucasian family with hyperproinsulinemia was identified through unexplained hyperinsulinemia in a normal volunteer participating in a metabolic study. High pressure liquid chromatography analysis of fasting plasma revealed a major peak eluting close to the position of proinsulin. Direct sequencing of the proinsulin gene exon 3 showed a heterozygous point mutation (CGT-->CAT) resulting in the substitution of Arg-->His in position 65 (corresponding to the AC cleavage site) in the index case, his mother, and his maternal grandmother. Using specific enzyme-linked immunosorbent assay methods to quantify insulin and proinsulin (including its conversion intermediates), the impact of this mutation on B cell secretion and glucose tolerance was studied. All affected subjects had normal oral glucose tolerance. In the basal state and after oral glucose administration, their proinsulin responses were immense, but intact insulin responses were slightly reduced. However, when calculating insulin bioactivity by assuming 9% activity for mutant Arg65-->His proinsulin, responses in affected subjects were comparable to those in normal subjects. In conclusion, our data demonstrate hyperproinsulinemia in a three-generation Caucasian family due to heterozygous mutant Arg65-->His proinsulin. This was not associated with impaired glucose tolerance. These results suggest that this mutation in the heterozygous state per se does not affect glucose tolerance and that the biological activity of mutant proinsulin contributes to glucose homeostasis in this family. The association of the same mutation with impaired glucose tolerance or diabetes in previous studies may be the result of selection bias or associated conditions (e.g. the genetic background of the kindreds examined).

Repression of transcriptional activity by heterologous KRAB domains present in zinc finger proteins.

We report the characterization of three novel members of the KRAB-domain containing C2-H2 zinc finger family (ZNF133, 136 and 140). KRAB (Krüppel-associated box) is an evolutionarily conserved protein domain found N-terminally with respect to the zinc finger repeats that encodes the DNA binding domain. ZNF133 and ZNF140 have both the KRAB A- and KRAB B-boxes present at their N-terminus, whereas ZNF136 contains only the KRAB A-box. We have previously demonstrated that the KRAB domains derived from ZNF133 and ZNF140 are potent transcriptional repression domains [Margolin et al. (1994) Proc. Natl. Acad. Sci. USA 91, 4509-4513]. The KRAB domain from ZNF136, containing only subdomain A, is a considerable weaker suppression domain; however, when fused to the heterologous KRAB B subdomain of ZNF10 (KOX1) the two subdomains from a KRAB domain which induces repression as potently as previously reported KRAB domains.

Cloning and expression of cytokine-inducible nitric oxide synthase cDNA from rat islets of Langerhans.

An inducible nitric oxide (NO) synthase isoform (iNOS) is specifically induced in the beta-cells of interleukin (IL)-1 beta-exposed rat islets, suggesting a role for NO in the pathogenesis of type I diabetes. The aim of this study was to clone and characterize iNOS cDNA from cytokine-exposed islets. Neither NO production nor iNOS transcription could be detected in rat islets or in rat insulinoma RIN-5AH beta-cells cultured in the absence of cytokines. Addition of IL-1 beta alone or in combination with tumor necrosis factor-alpha induced a concentration- and time-dependent expression of the iNOS gene and associated NO production (measured as nitrite) from both islets and RIN cells. iNOS transcripts were cloned by reverse transcriptase-polymerase chain reaction from the cytokine-exposed rat islets and RIN cells, and DNA sequence analysis revealed a near 100% identity to the recently published iNOS cDNA cloned from cytokine-exposed rat hepatocytes and smooth muscle cells. Recombinant rat islet iNOS was transiently and stably expressed in human kidney 293 fibroblasts, and the high enzymatic activity was inhibited by addition of the L-arginine analogs, N omega-nitro-L-arginine methyl ester and aminoguanidine. Two-dimensional gel electrophoresis revealed the recombinant iNOS as a series of spots with the expected molecular mass of 131 kDa and pI values in the range of 6.8 to 7.0. In conclusion, the IL-1 beta-induced iNOS cloned and expressed from rat islets and RIN cells is encoded by the same transcript as the iNOS induced in other cell types.

Isolation and fine mapping of 16 novel human zinc finger-encoding cDNAs identify putative candidate genes for developmental and malignant disorders.

We have isolated and chromosomally fine-mapped 16 novel genes belonging to the human zinc finger Krüppel family (ZNF131-140, 142, 143, 148, 151, 154, and 155), including 1 of the GLI type (ZNF143) and 3 containing a KRAB (Krüppel-associated box) segment (ZNF133, 136, and 140). Based on their map position, several of these ZNF genes are putative candidate genes for both developmental and malignant disorders: ZNF138, ZNF139, and ZNF143 were localized to 7q11.2, 7q21.3-q22.1, and 11p15.3-p15.4, regions involved in deletions and/or translocations associated with Williams syndrome, split hand and foot disease (SHFD1), and Beckwith-Wiedemann syndrome, respectively. ZNF133 was localized to 20p11.2, close to, but probably distinct from, the region deleted in Alagille syndrome. Zinc finger genes mapping to regions commonly deleted in solid tumors included ZNF132, 134, 135, 137, 154, and 155, all located on 19q13 (thyroid adenoma), and ZNF151, at 1p36.1-p36.2 (neuroblastoma, colon cancer, and other tumors). In addition, several of the ZNFs mapped to regions implicated in recurrent chromosomal rearrangements in hematological malignancies (ZNF139, 7q21.3-q22.1; ZNF148, 3q21-q22; ZNF151, 1p36.1-p36.2). The study indicates that the number of ZNF genes in human is large and that systematic isolation and mapping of ZNF genes is a straightforward approach for the identification of novel candidate disease genes.

Negative cooperativity in the insulin-like growth factor-I receptor and a chimeric IGF-I/insulin receptor.

Insulin and insulin-like growth factor-I (IGF-I) share a spectrum of metabolic and growth-promoting effects, mediated through homologous receptors that belong to the tyrosine kinase family. The dissociation rate of insulin from its receptor is affected by negative cooperativity, i.e. accelerates with increased receptor occupancy. The dose-response curve for the acceleration of tracer dissociation by unlabeled insulin has a distinct bell-shaped curve, with a progressive slowing down at insulin concentrations greater than 100 nM. The kinetics of the IGF-I interaction with its receptor has not been studied in such detail. In the present work, we report that while the IGF-I receptor exhibits negative cooperativity like the insulin receptor, the concentration dependence of the dissociation kinetics is distinct from that of native human insulin by not being bell-shaped, but monophasic like that of insulin analogues mutated at the hexamer-forming surface; it is changed to an insulin-type curve by substitution of IGF-I receptor's sequence including residues 382-565 with the homologous insulin receptor domain. The data suggest that like insulin, IGF-I has a bivalent binding mode and crosslinks two distinct areas of the two alpha subunits that are close, but distinct from the equivalent insulin receptor binding sites.

Krüppel-associated boxes are potent transcriptional repression domains.

The Krüppel-associated box (KRAB) is a highly conserved, 75-aa region containing two predicted amphipathic alpha-helices. The KRAB domain is present in the amino-terminal regions of more than one-third of all Krüppel-class Cys2His2 zinc finger proteins and is conserved from yeast to man; however, its function is unknown. Here it is shown that the KRAB domain functions as a DNA binding-dependent transcriptional repressor when fused to a heterologous DNA-binding domain from the yeast GAL4 protein. A 45-aa segment containing one of the predicted KRAB amphipathic helices was necessary and sufficient for repression. Amino acid substitutions in the predicted helix abolished the repression function. These results assign a function, transcriptional repression, to the highly conserved KRAB box and define a minimal repression domain which may aid in identifying mechanisms of repression.