GPR40 gene expression in human pancreas and insulinoma

To assess gene expression of a membrane-bound G-protein-coupled fatty acid receptor, GPR40, in the human pancreas and islet cell tumors obtained at surgery were analyzed. The mRNA level of the GPR40 gene in isolated pancreatic islets was approximately 20-fold higher than that in the pancreas, and the level was comparable to or rather higher than that of the sulfonylurea receptor 1 gene, which is known to be expressed abundantly in human pancreatic beta cells. A large amount of GPR40 mRNA was detected in tissue extracts from two cases of insulinoma, whereas the expression was undetectable in glucagonoma or gastrinoma. The present study demonstrates that GPR40 mRNA is expressed predominantly in pancreatic islets in humans and that GPR40 mRNA is expressed solely in human insulinoma among islet cell tumors. These results indicate that GPR40 is probably expressed in pancreatic beta cells in the human pancreas.     

Control of Cell pH in the T84 Colon Cell Line

Cell pH regulation was investigated in the T84 cell line derived from epithelial colon cancer. Cell pH was measured by ratiometric fluorescence microscopy using the fluorescent probe BCECF. Basal pH was 7.17 ± 0.023 (n= 48) in HEPES Ringer. After acidification by an ammonium pulse, cell pH recovered toward normal at a rate of 0.13 ± 0.011 pH units/min in the presence of Na⁺, but in the absence of this ion or after treatment with 0.1 mm hexamethylene amiloride (HMA) no significant recovery was observed, indicating absence of Na⁺ independent H⁺ transport mechanisms in HEPES Ringer. In CO₂/HCO⁻ ₃ Ringer, basal cell pH was 7.21 ± 0.020 (n= 35). Changing to HEPES Ringer, a marked alkalinization was observed due to loss of CO₂, followed by return to the initial pH at a rate of −0.14 ± 0.012 (n= 8) pH/min; this return was retarded or abolished in the absence of Cl⁻ or after addition of 0.2 mm DIDS, suggesting extrusion of bicarbonate by Cl⁻/HCO⁻ ₃ exchange. This exchange was not Na⁺ dependent. When Na⁺ was added to cells incubated in 0 Na⁺ Ringer while blocking Na⁺/H⁺ exchange by HMA, cell alkalinization by 0.19 ± 0.04 (n= 11) pH units was observed, suggesting the presence of Na⁺/HCO⁻ ₃ cotransport carrying HCO⁻ ₃ into these cells, which was abolished by DIDS. These experiments, thus, show that Na⁺/H⁺ and Cl⁻/HCO⁻ ₃ exchange and Na⁺/HCO⁻ ₃ cotransport participate in cell pH regulation in T84 cells. Received: 3 April 2000/Revised: 22 June 2000     

Proton-sensing and lysolipid-sensitive G-protein-coupled receptors: a novel type of multi-functional receptors

OGR1, GPR4, G2A, and TDAG8 share 40% to 50% homology with each other and seem to form a family of GPCRs. They have been described as receptors for lipid molecules such as sphingosylphosphorylcholine, lysophosphatidylcholine, and psychosine. Recent studies, however, have revealed that these receptors also sense extracellular protons or pH through histidine residues of receptors and stimulate a variety of intracellular signaling pathways through several species of hetero-trimeric G-proteins, including G_s, G_i, G_q, and G_12/13. Thus, this family of GPCR seems to recognize both lipid molecules and protons as ligands. Although our knowledge of proton-sensing and lysolipid-sensitive GPCRs is preliminary, the receptor levels and ligand levels especially protons are both sensitively modulated in response to a variety of microenvironmental changes. These results suggest a multiple role of proton-sensing GPCRs in a variety of physiological and pathophysiological states.     

NMR measurements of the diffusional permeability of water in cultured colonic epithelial cancer cells

The water residence time and diffusional water permeability in colonic epithelial T84 cancer cells was measured using (1)H NMR spectroscopy; the values estimated were 35.2+/-2.8 ms and (7.4+/-0.6)x10(-3)cms(-1), respectively. Water permeability was inhibited to approximately 10% of its original value by the mercurial diuretic, p-chloromercuribenzenesulfonate (PCMBS; 1mM), and fully restored by dithiothreitol (DTT; 1mM). The permeability was also inhibited reversibly to approximately 55%, by extracellular glibenclamide (1mM), an inhibitor of some ATP-binding cassette (ABC) transporters, including the cystic fibrosis transmembrane conductance regulator (CFTR). Addition of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IMBX; 0.1-1mM) and the adenylate cyclase activator, forskolin (0.1-1mM) did not alter water permeability. It is concluded that in T84 cells water diffuses through the membrane lipid bilayer and via channels that are inhibited by PCMBS, including the channels that are known to be inhibited by glibenclamide.     

GPR80/99, proposed to be the P2Y<Subscript>15</Subscript> receptor activated by adenosine and AMP,is not a P2Y receptor

The orphan receptor GPR80 (also called GPR99) was recently reported to be the P2Y₁₅ receptor activated by AMP and adenosine and coupled to increases in cyclic AMP accumulation and intracellular Ca²⁺ mobilization (Inbe et al. J Biol Chem 2004; 279: 19790–9). However, the cell line (HEK293) used to carry out those studies endogenously expresses A_2A and A_2B adenosine receptors as well as multiple P2Y receptors, which complicates the analysis of a potential P2Y receptor. To determine unambiguously whether GPR80 is a P2Y receptor subtype, HA-tagged GPR80 was either stably expressed in CHO cells or transiently expressed in COS-7 and HEK293 cells, and cell surface expression was verified by radioimmunoassay (RIA). COS-7 cells overexpressing GPR80 showed a consistent twofold increase in basal inositol phosphate accumulation. However, neither adenosine nor AMP was capable of promoting accumulation of either cyclic AMP or inositol phosphates in any of the three GPR80-expressing cells. A recent paper (He et al. Nature 2004; 429: 188–93) reported that GPR80 is a Gq-coupled receptor activated by the citric acid cycle intermediate, -ketoglutarate. Consistent with this report, -ketoglutarate promoted inositol phosphate accumulation in CHO and HEK293 cells expressing GPR80, and pretreatment of GPR80-expressing COS-7 cells with glutamate dehydrogenase, which converts -ketoglutarate to glutamate, decreased basal levels of inositol phosphates. Taken together, these data demonstrate that GPR80 is not activated by adenosine, AMP or other nucleotides, but instead is activated by -ketoglutarate. Therefore, GPR80 is not a new member of the P2Y receptor family.     

A-kinase anchor protein 84/121 are targeted to mitochondria and mitotic spindles by overlapping amino-terminal motifs

A-kinase anchor proteins (AKAPs) assemble multi-enzyme signaling complexes in proximity to substrate/effector proteins, thus directing and amplifying membrane-generated signals. S-AKAP84 and AKAP121 are alternative splicing products with identical NH(2) termini. These AKAPs bind and target protein kinase A (PKA) to the outer mitochondrial membrane. Tubulin was identified as a binding partner of S-AKAP84 in a yeast two-hybrid screen. Immunoprecipitation and co-sedimentation experiments in rat testis extracts confirmed the interaction between microtubules and S-AKAP84. In situ immunostaining of testicular germ cells (GC2) shows that AKAP121 concentrates on mitochondria in interphase and on mitotic spindles during M phase. Purified tubulin binds directly to S-AKAP84 but not to a deletion mutant lacking the mitochondrial targeting domain (MT) at residues 1-30. The MT is predicted to form a highly hydrophobic alpha-helical wheel that might also mediate interaction with tubulin. Disruption of the wheel by site-directed mutagenesis abolished tubulin binding and reduced mitochondrial attachment of an MT-GFP fusion protein. Some MT mutants retain tubulin binding but do not localize to mitochondria. Thus, the tubulin-binding motif lies within the mitochondrial attachment motif. Our findings indicate that S-AKAP84/AKAP121 use overlapping targeting motifs to localize signaling enzymes to mitochondrial and cytoskeletal compartments.     

Quercetin-3-oleoyl derivatives as new GPR40 agonists: Molecular docking studies and functional evaluation

The G-protein-coupled receptor 40 (GPR40) is an attractive molecular target for the treatment of type 2 diabetes mellitus. Previously, based on the natural oleic acid substrate, an exogenous ligand for this receptor, named AV1, was synthesized. In this context, here we validated the activity of AV1 as a full agonist, while the corresponding catechol analogue, named AV2, was investigated for the first time. The ligand-protein interaction between this new molecule and the receptor was highlighted in the lower portion of the GPR40 groove that generally accommodates DC260126. The functional assays performed have demonstrated that AV2 is a suitable GPR40 partial agonist, showing a therapeutic potential and representing a useful tool in the management of type 2 diabetes.     

Construction of a Tra? deletion mutant of pAgK84 to safeguard the biological control of crown gall

Summary Agrobacterium radiobacter strain K84 is used commercially for the biological control of crown gall. It contains the conjugative plasmid pAgK84, which encodes the synthesis of agrocin 84, an antibiotic that inhibits many pathogenic agrobacteria. A breakdown of control is threatened by the transfer of pAgK84 to pathogens, which then become resistant to agrocin 84. A mutant of pAgK84 with a 5.9-kb deletion overlapping the transfer (Tra) region was constructed using recombinant DNA techniques. The BamHI fragment B1 which covers most of the Tra region was cloned in pBR325 and its internal EcoRI fragments D1 and H, which overlap the Tra region, were removed, leaving 3.7 kb and 0.5 kb of pAgK84 on either side of the deletion. The latter was increased to 3.3 kb by adding EcoRI fragment D2 from a BamHI fragment C clone. The modified pBR325 clone was mobilized into Agrobacterium strain NT1 harbouring pAgK84 with a Tn5 insertion just outside the Tra region but covered by the deletion. A Tra⁺ cointegrate was formed between the Tn5-insertion derivative and the pBR325-based deletion construct by homologous recombination. The cointegrate was transferred by conjugation to a derivative of strain K84 lacking pAgK84, in which a second recombination event generated a stable deletion-mutant by deletion-marker exchange. The resultant new strain of A. radiobacter, designated K1026, shows normal agrocin 84 production. Mating experiments show that the mutant plasmid, designated pAgK1026, is incapable of conjugal transfer at a detectable frequency.     

Whirlin and PDZ Domain-containing 7 (PDZD7) Proteins Are Both Required to Form the Quaternary Protein Complex Associated with Usher Syndrome Type 2

Usher syndrome (USH) is the leading genetic cause of combined hearing and vision loss. Among the three USH clinical types, type 2 (USH2) occurs most commonly. USH2A, GPR98, and WHRN are three known causative genes of USH2, whereas PDZD7 is a modifier gene found in USH2 patients. The proteins encoded by these four USH genes have been proposed to form a multiprotein complex, the USH2 complex, due to interactions found among some of these proteins in vitro, their colocalization in vivo, and mutual dependence of some of these proteins for their normal in vivo localizations. However, evidence showing the formation of the USH2 complex is missing, and details on how this complex is formed remain elusive. Here, we systematically investigated interactions among the intracellular regions of the four USH proteins using colocalization, yeast two-hybrid, and pull-down assays. We show that multiple domains of the four USH proteins interact among one another. Importantly, both WHRN and PDZD7 are required for the complex formation with USH2A and GPR98. In this USH2 quaternary complex, WHRN prefers to bind to USH2A, whereas PDZD7 prefers to bind to GPR98. Interaction between WHRN and PDZD7 is the bridge between USH2A and GPR98. Additionally, the USH2 quaternary complex has a variable stoichiometry. These findings suggest that a non-obligate, short term, and dynamic USH2 quaternary protein complex may exist in vivo. Our work provides valuable insight into the physiological role of the USH2 complex in vivo and informs possible reconstruction of the USH2 complex for future therapy.