cAMP Accumulation In COS-7 Cells Research Articles

Glucose-dependent insulinotropic polypeptide receptor antagonist treatment causes a reduction in weight gain in ovariectomised high fat diet-fed mice.

Background and purposeThe incretin hormone, gastric inhibitory peptide/glucose‐dependent insulinotropic polypeptide (GIP), secreted by the enteroendocrine K‐cells in the proximal intestine, may regulate lipid metabolism and adiposity, but its exact role in these processes is unclear.Experimental approachWe characterized in vitro and in vivo antagonistic properties of a novel GIP analogue, mGIPAnt‐1. We further assessed the in vivo pharmacokinetic profile of this antagonist, as well as its ability to affect high‐fat diet (HFD)‐induced body weight gain in ovariectomised mice during an 8‐week treatment period.Key resultsmGIPAnt‐1 showed competitive antagonistic properties to the GIP receptor in vitro as it inhibited GIP‐induced cAMP accumulation in COS‐7 cells. Furthermore, mGIPAnt‐1 was capable of inhibiting GIP‐induced glucoregulatory and insulinotropic effects in vivo and has a favourable pharmacokinetic profile with a half‐life of 7.2 h in C57Bl6 female mice. Finally, sub‐chronic treatment with mGIPAnt‐1 in ovariectomised HFD mice resulted in a reduction of body weight and fat mass.Conclusion and ImplicationsmGIPAnt‐1 successfully inhibited acute GIP‐induced effects in vitro and in vivo and sub‐chronically induces resistance to HFD‐induced weight gain in ovariectomised mice. Our results support the development of GIP antagonists for the therapy of obesity.

Histamine Modulates Isoproterenol Efficacy at the β2 Adrenoceptor: Inferences Regarding Allosteric Modulation by Imidazole-Containing Compounds

In the last few years, evidence has been mounting of the possible allosteric modulation of second messenger formation on β2-adrenoceptors (β2ARs) induced by non-arylethylamine compounds. We herein addressed this issue by carrying out functional and molecular modeling studies to explore the possibility that imidazole-containing compounds (ICCs) affect the formation and accumulation of cAMP by mediating β2AR activation. Results show that histamine (an ICC) had no effect on basal cAMP accumulation in COS-7 cells transfected with the human β2AR (10.4 ± 1.1 pmol/mg protein), but significantly augmented the receptor response to the agonist isoproterenol (137 ± 7% of controls, EC50 10.5 μM, pEC50 5.87 ± 0.06). Moreover, histamine (10 μM) did not affect the isoproterenol inhibition of 3H]-dihydroalprenolol binding to hβ2ARs in membranes of transfected COS-7 cells. Q-site Finder program and molecular docking studies identified possible sites of interaction for ICCs on the β2AR, but the estimated affinities were lower than those reported for well-known β2AR ligands on the orthosteric site. On the basis of the present experimental and theoretical data, we postulate that direct ICC-hβ2AR interactions can allosterically modulate agonist-induced receptor activation. Implications of these findings for drug design are discussed.

Inhibition of Heterotrimeric G Protein Signaling by a Small Molecule Acting on Gα Subunit

The simultaneous activation of many distinct G protein-coupled receptors (GPCRs) and heterotrimeric G proteins play a major role in various pathological conditions. Pan-inhibition of GPCR signaling by small molecules thus represents a novel strategy to treat various diseases. To better understand such therapeutic approach, we have characterized the biomolecular target of BIM-46187, a small molecule pan-inhibitor of GPCR signaling. Combining bioluminescence and fluorescence resonance energy transfer techniques in living cells as well as in reconstituted receptor-G protein complexes, we observed that, by direct binding to the Galpha subunit, BIM-46187 prevents the conformational changes of the receptor-G protein complex associated with GPCR activation. Such a binding prevents the proper interaction of receptors with the G protein heterotrimer and inhibits the agonist-promoted GDP/GTP exchange. These observations bring further evidence that inhibiting G protein activation through direct binding to the Galpha subunit is feasible and should constitute a new strategy for therapeutic intervention.

CONDITIONAL STIMULATION OF TYPE V AND VI ADENYLYL CYCLASES BY G PROTEIN βγ SUBUNITS

In a yeast two hybrid screen of mouse brain cDNA library, using the N-terminus of human type V adenylyl cyclase (hACV) as bait, we identified G protein β2 subunit as an interacting partner. Additional yeast two hybrid assays showed that the Gβ1 subunit also interacts with the N-terminus of hACV and human type VI adenylyl cyclase (hACVI). In-vitro adenylyl cyclase activity assays using membranes of Sf9 cells expressing hACV or hACVI showed that Gβγ subunits enhance the activity of these enzymes in presence of Gαs or forskolin. Deletion of residues 77–151, but not 1–76, in the N-terminus of hACVI obliterated the ability of Gβγ subunits to stimulate the enzyme. Likewise, activities of the recombinant soluble forms of ACV and ACVI, which lack the N-termini, were not enhanced by Gβγ subunits. Transfection of the C terminus of G protein receptor kinase 2 to sequester endogenous Gβγ subunits attenuated the ability of isoproterenol to increase cAMP accumulation in COS-7 cells overexpressing hACVI even when Gi was inactivated by pertussis toxin. Therefore, we conclude that the N-termini of human hACV and hACVI are necessary for their interactions with, and regulation by Gβγ subunits both in-vitro and in intact cells. Moreover, Gβγ subunits derived from source(s) other than Gi are necessary for the full activation of hACVI by isoproterenol in intact cells. Supported by NIH grants HL59679 and GM073181 to TBP and GM060419 to CWD.

Conditional Stimulation of Type V and VI Adenylyl Cyclases by G Protein βγ Subunits

In a yeast two-hybrid screen of mouse brain cDNA library, using the N-terminal region of human type V adenylyl cyclase (hACV) as bait, we identified G protein beta2 subunit as an interacting partner. Additional yeast two-hybrid assays showed that the Gbeta(1) subunit also interacts with the N-terminal segments of hACV and human type VI adenylyl cyclase (hACVI). In vitro adenylyl cyclase (AC) activity assays using membranes of Sf9 cells expressing hACV or hACVI showed that Gbetagamma subunits enhance the activity of these enzymes provided either Galpha(s) or forskolin is present. Deletion of residues 77-151, but not 1-76, in the N-terminal region of hACVI obliterated the ability of Gbetagamma subunits to conditionally stimulate the enzyme. Likewise, activities of the recombinant, engineered, soluble forms of ACV and ACVI, which lack the N termini, were not enhanced by Gbetagamma subunits. Transfection of the C terminus of G protein receptor kinase 2 to sequester endogenous Gbetagamma subunits attenuated the ability of isoproterenol to increase cAMP accumulation in COS-7 cells overexpressing hACVI even when G(i) was inactivated by pertussis toxin. Therefore, we conclude that the N termini of human hACV and hACVI are necessary for interactions with, and regulation by, Gbetagamma subunits both in vitro and in intact cells. Moreover, Gbetagamma subunits derived from a source(s) other than G(i) are necessary for the full activation of hACVI by isoproterenol in intact cells.

VIP provides cellular protection through a specific splice variant of the PACAP receptor: A new neuroprotection target

Vasoactive intestinal peptide (VIP) was known to provide neuroprotection. Three VIP receptors have been cloned: VPAC1, VPAC2 and PAC1. A specific splice variant of PAC1 in the third cytoplasmatic loop, hop2, was implicated in VIP-related neuroprotection. We aimed to clone the hop2 splice variant, examine its affinity to VIP and investigate whether it mediates the VIP-related neuroprotective activity. The PAC1 cDNA was cloned from rat cerebral astrocytes. Using genetic manipulation the hop2 splice variant was obtained, then inserted into an expression vector and transfected into COS-7 cells that were used for binding assays. Results showed that VIP bound the cloned hop2 splice variant. Stearyl-neurotensin 6–11 VIP 7–28 (SNH), an antagonist for VIP, was also found to bind hop2. In addition, VIP protected COS-7 cells expressing hop2 from oxidative stress. Parallel assays demonstrated that VIP increased cAMP accumulation in COS-7 cells expressing hop2. These results support the hypothesis that hop2 mediates the cytoprotective effects attributed to VIP.

Activation of the β2-Adrenergic Receptor Involves Disruption of an Ionic Lock between the Cytoplasmic Ends of Transmembrane Segments 3 and 6

The movements of transmembrane segments (TMs) 3 and 6 at the cytoplasmic side of the membrane play an important role in the activation of G-protein-coupled receptors. Here we provide evidence for the existence of an ionic lock that constrains the relative mobility of the cytoplasmic ends of TM3 and TM6 in the inactive state of the beta(2)-adrenergic receptor. We propose that the highly conserved Arg-131(3.50) at the cytoplasmic end of TM3 interacts both with the adjacent Asp-130(3.49) and with Glu-268(6.30) at the cytoplasmic end of TM6. Such a network of ionic interactions has now been directly supported by the high-resolution structure of the inactive state of rhodopsin. We hypothesized that the network of interactions would serve to constrain the receptor in the inactive state, and the release of this ionic lock could be a key step in receptor activation. To test this hypothesis, we made charge-neutralizing mutations of Glu-268(6.30) and of Asp-130(3.49) in the beta(2)-adrenergic receptor. Alone and in combination, we observed a significant increase in basal and pindolol-stimulated cAMP accumulation in COS-7 cells transiently transfected with the mutant receptors. Moreover, based on the increased accessibility of Cys-285(6.47) in TM6, we provide evidence for a conformational rearrangement of TM6 that is highly correlated with the extent of constitutive activity of the different mutants. The present experimental data together with the recent high-resolution structure of rhodopsin suggest that ionic interactions between Asp/Glu(3.49), Arg(3.50), and Glu(6.30) may constitute a common switch governing the activation of many rhodopsin-like G-protein-coupled receptors.

Juxtamembrane Region of the Amino Terminus of the Corticotropin Releasing Factor Receptor Type 1 Is Important for Ligand Interaction

The functional properties of the amino terminus (NT) of the corticotropin releasing factor (CRF) receptor type 1 (R1) were studied by use of murine (m) CRFR1 and rat (r) parathyroid hormone (PTH)/parathyroid hormone-related peptide receptor (PTH1R) chimeras. The chimeric receptor CXP, in which the NT of mCRFR1 was annealed to the TMs of PTH1R, and the reciprocal hybrid, PXC, bound radiolabeled analogues of sauvagine and PTH(3--34), respectively. Neither hybrid bound radiolabeled CRF or PTH(1--34). CRF and PTH(1--34) weakly stimulated intracellular cAMP accumulation in COS-7 cells transfected with PXC and CXP, respectively. Thus the NT is required for ligand binding and the TMs are required for agonist-stimulated cAMP accumulation. Replacing individual intercysteine segments of PXC with their mCRFR1 counterparts did not rescue CRF or sauvagine radioligand binding or stimulation of cAMP accumulation. Replacement of residues 1--31 of mCRFR1 with their PTH1R counterparts resulted in a chimeric receptor, PEC, which had normal CRFR1 functional properties. In addition, a series of chimeras (F1PEC--F6PEC) were generated by replacement of the NT intercysteine residues of PEC with their PTH1R counterparts. Only F1PEC, F2PEC, and F3PEC showed detectable CRF and sauvagine radioligand binding. All of the PEC chimeras except F5PEC increased cAMP accumulation. These data indicate that the Cys(68)(-)Glu(109) domain is important for binding and that the Cys(87)(-)Cys(102) region plays an important role in CRFR1 activation.

Tissue-specific transcription start sites and alternative splicing of the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor gene: a new PTH/PTHrP receptor splice variant that lacks the signal peptide.

The PTH/PTHrP receptor gene is expressed in bone and kidney as well as in many other tissues. Using primer extension followed by rapid cloning of amplified complementary DNA ends, we have isolated new PTH/PTHrP receptor complementary DNAs with different splicing patterns and have characterized a new upstream transcription start site. Three 5' nontranslated exons, U3, U2 and U1, located 4.8, 2.5, and 1.2 kb upstream of the exon that encodes the putative signal peptide of the classical receptor (exon S), have been characterized. Four types of splicing patterns were recognized. Type I splicing pattern is transcribed from exon U1 and is spliced to exons S and E1; this pattern was found in most tissues tested. Types II, III, and IV splicing patterns are transcribed from exon U3 and have a restricted tissue distribution. Type II splice pattern, containing exons U3, U2, and S and type III splicing pattern, containing exon U3, U2, and E1 (skipping exon S), was found only in kidney. Type IV splice pattern, containing exon U3 and S was found both in kidney and ovary. Because the type III splice variant skips exon S, translation of this splice variant initiates at a different AUG codon. The type III splice variant was weakly expressed on the cell surface of COS-7 cells, as assessed by double antibody binding assay, and no detectable ligand binding was observed on intact cells. The type III splice variant, however, increased cAMP accumulation in COS-7 cells when challenged with PTH(1-34), PTH(1-84) and hPTHrP(1-36) with EC50s that are similar to those observed in COS-7 cells expressing the type I variant but with a maximum stimulation that was lower than that observed in COS-7 cells expressing the type I variant. These data indicate low levels of cell surface expression of the type III splice variant. Treatment of COS-7 cells with tunicamycin decreased the size of the type I splice variant from a broad band of 85 kDa to a compact band of about 60 kDa. The type III splice variant did not change in size in COS-7 cells treated with tunicamycin, indicating that the type III splice variant did not undergo any glycosylation step. In conclusion, the PTH/PTHrP receptor gene uses alternate promoters in a tissue-specific manner that results in several tissue-specific alternatively spliced transcripts. One of these transcripts, the type III splice variant, is expressed in kidney and lacks the signal peptide.

Tissue-Specific Transcription Start Sites and Alternative Splicing of the Parathyroid Hormone (PTH)/PTH-Related Peptide (PTHrP) Receptor Gene: A New PTH/PTHrP Receptor Splice Variant that Lacks the Signal Peptide

The PTH/PTHrP receptor gene is expressed in bone and kidney as well as in many other tissues. Using primer extension followed by rapid cloning of amplified complementary DNA ends, we have isolated new PTH/PTHrP receptor complementary DNAs with different splicing patterns and have characterized a new upstream transcription start site. Three 5′ nontranslated exons, U3, U2 and U1, located 4.8, 2.5, and 1.2 kb upstream of the exon that encodes the putative signal peptide of the classical receptor (exon S), have been characterized. Four types of splicing patterns were recognized. Type I splicing pattern is transcribed from exon U1 and is spliced to exons S and E1; this pattern was found in most tissues tested. Types II, III, and IV splicing patterns are transcribed from exon U3 and have a restricted tissue distribution. Type II splice pattern, containing exons U3, U2, and S and type III splicing pattern, containing exon U3, U2, and E1 (skipping exon S), was found only in kidney. Type IV splice pattern, containing exon U3 and S was found both in kidney and ovary. Because the type III splice variant skips exon S, translation of this splice variant initiates at a different AUG codon. The type III splice variant was weakly expressed on the cell surface of COS-7 cells, as assessed by double antibody binding assay, and no detectable ligand binding was observed on intact cells. The type III splice variant, however, increased cAMP accumulation in COS-7 cells when challenged with PTH(1–34), PTH(1–84) and hPTHrP(1–36) with EC50s that are similar to those observed in COS-7 cells expressing the type I variant but with a maximum stimulation that was lower than that observed in COS-7 cells expressing the type I variant. These data indicate low levels of cell surface expression of the type III splice variant. Treatment of COS-7 cells with tunicamycin decreased the size of the type I splice variant from a broad band of 85 kDa to a compact band of about 60 kDa. The type III splice variant did not change in size in COS-7 cells treated with tunicamycin, indicating that the type III splice variant did not undergo any glycosylation step. In conclusion, the PTH/PTHrP receptor gene uses alternate promoters in a tissue-specific manner that results in several tissue-specific alternatively spliced transcripts. One of these transcripts, the type III splice variant, is expressed in kidney and lacks the signal peptide.

GRK2 and beta-arrestin 1 as negative regulators of thyrotropin receptor-stimulated response.

Arrestins are regulatory proteins for a number of G-coupled receptors. The binding of arrestin to receptor phosphorylated by G protein-coupled receptor kinase (GRK) quenches the activation of the G protein, thus resulting in receptor homologous desensitization. We have previously shown that the levels of beta-arrestin1 are regulated by intracellular cAMP and proposed that this may represent one homeostatic mechanism with which to regulate some cellular responses. To test this hypothesis, we focused on the TSH receptor using a rat thyroid cell line, FRTL5. We found that beta-arrestin1 is the only detectable isoform of arrestin expressed in FRTL5 and that its expression is regulated by TSH. To investigate the possible role of GRK2/beta-arrestin1 machinery in the mechanism of TSH receptor homologous desensitization, we used a cotransfection approach. The TSH-induced cAMP accumulation in COS7 cells transfected with TSH receptor was reduced by 35-45% when cotransfected with GRK2 and/or beta-arrestin1, indicating that the TSH receptor can be regulated by a GRK/arrestin mechanism. This raised the hypothesis that TSH increases the levels of beta-arrestin1, which in turn could regulate the TSH stimulation. To test this point a FRTL5-derived cell line overexpressing beta-arrestin1 was generated. In these cells the TSH-stimulated cAMP accumulation and, more importantly, the mitogenic activity were substantially blunted. Our results show that TSH receptor-stimulated cAMP accumulation and cell proliferation can be controlled by a GRK2/beta-arrestin1 mechanism.

Molecular cloning of a type A chicken corticotropin-releasing factor receptor with high affinity for urotensin I.

The hypothalamic-pituitary-adrenal axis is an essential physiological system in many species. CRF, the major neuropeptide regulating ACTH secretion, is highly conserved in its primary sequence. Evolutionary conservation of the CRF sequence suggests that the CRF receptor (CRF-R) complementary DNA and examined its properties. The avian CRF-R complementary DNA encodes a 420-amino acid protein that is 87-88% identical to those of human, rat, and mouse. Most sequence divergence occurs in the putative signal peptide and the extracellular amino-terminus of the receptor. Five additional amino acids are inserted in the amino-terminus of the cCRF-R. When expressed in COS-7 cells, the cCRF-R binds the CRF and urotensin I radioligands with high affinities. Urotensin I competes for binding to the chicken CRF-R, expressed in COS-7 cells, with an apparent affinity 20 times higher than that of CRF. Both urotensin I and sauvagine were more effective in stimulating cAMP accumulation in COS-7 cells transfected with the cCRF-R than CRF. The effects of CRF and urotensin I on inositol phosphate accumulation were also tested. Urotensin I was an effective as CRF in stimulating inositol phosphate accumulation in COS-7 cells transfected with the cCRF-R. These data suggest that the sequence of the CRF-R is highly conserved from avian to mammalian species and that, despite its high sequence homology to the type A mammalian CRF-R, the ligand binding properties of cCRF-R are similar to those of the type B CRF-R i.e. a higher affinity for urotensin I than for CRF.

Signaling properties of mouse and human corticotropin-releasing factor (CRF) receptors: decreased coupling efficiency of human type II CRF receptor.

CRF is the primary neuroregulator of the function of the hypothalamic-pituitary-adrenal axis. We have recently cloned a mouse CRF receptor (mCRF-R) complementary DNA (cDNA) from an AtT-20 cell cDNA library by polymerase chain reaction. To compare the functions of mCRF-R to those of the human type I and type II CRF receptors (hCRF-RI and hCRF-RII), cDNAs were cloned into the expression vector pcDNA1 and transfected into COS-7 cells. CRF binding and CRF-stimulated cAMP accumulation as well as phosphoinositide hydrolysis were measured. Scatchard analysis of the binding of 125I-labeled [Tyr0]r/hCRF ([125I]CRF) to COS-7 cells expressing mCRF-R and hCRF-RI cDNAs revealed the same apparent Kd (9 nM). In contrast, the apparent binding Kd for hCRF-RII was 20 nM CRF. Maximal stimulatory concentrations (1 microM) of rat/human CRF-(1-41) (r/hCRF) increased cAMP accumulation in COS-7 cells transfected with mCRF-R, hCRF-RI, and hCRF-RII cDNA plasmid (10 micrograms each) from basal values of 8-19 pmol/10(5) cells.15 min to 84 +/- 10, 87 +/- 16, and 45 +/- 16 pmol/10(5) cells.15 min, respectively. The EC50 values of r/hCRF-stimulated cAMP accumulation in COS-7 cells expressing mCRF-R and hCRF-RI cDNAs were similar at 0.4 +/- 0.2 and 0.7 +/- 0.2 nM, respectively. Conversely, the EC50 of r/hCRF-stimulated cAMP accumulation in hCRF-RII-transfected COS-7 cells was 47.5 +/- 18.9 nM. As the level of expression of hCRF-RII was lower than that of hCRF-RI, we compared r/hCRF-stimulated cAMP accumulation in COS-7 cells expressing low and high levels of hCRF-RI. The EC50 for r/hCRF-stimulated cAMP accumulation in COS-7 cells transfected with hCRF-RI did not change when receptor expression was varied by a factor of 1- to 8.4-fold. In contrast, the EC50 for r/hCRF-stimulated cAMP accumulation mediated by hCRF-RII was at least 100-fold higher than that mediated by the hCRF-RI in COS-7 cells, which suggests poor coupling between hCRF-RII and adenylate cyclase. Inositol phosphate (IP) levels were also determined in mCRF-R, hCRF-RI, and hCRF-RII cDNA-transfected COS-7 cells stimulated with increasing concentrations of r/hCRF. r/hCRF-stimulated IPs accumulation was dose dependent in COS-7 cells expressing mCRF-R and hCRF-RI using 100 and 1000 nM r/hCRF. Concentrations of 10 (or less) nM r/hCRF had no effect on IP generation. hCRF-RII did not mediate stimulation of IP even at 1000 nM r/hCRF.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino acids in the cloned mouse kappa receptor that are necessary for high affinity agonist binding but not antagonist binding

Opioids exert their biological effects through membrane-bound receptors. We have been interested in examining the domains and specific residues of the opioid receptors involved in ligand binding. In this report we demonstrate the critical roles of two highly conserved aspartate residues in the second and third transmembrane domains (TM2 and TM3) of the κ receptor in ligand binding. Two mutant receptors were generated in which the negatively charged aspartate residues residing in TM2 and TM3 were independently mutated to neutrally charged asparagines (D105N and D138N). These aspartate residues are absolutely required for high affinity κ-selective agonist binding. In addition, we exchanged the region encompassing TM4 and the second extracellular loop between the κ and δ opioid receptors. One of these chimeras, the κ receptor with TM4 and the δ second extracellular loop (κ/δ-2eloop), was unable to bind κ-selective agonists but bound antagonists and nonselective opioids in a manner identical to wild-type κ receptor. The ability of κ-selective agonists to inhibit forskolin-stimulated cAMP accumulation in COS-7 cells transfected with the κ/δ-2eloop chimera was completely abolished, consistent with the loss of binding observed. These results indicate that TM4 and/or the second extracellular loop are involved in κ-selective agonist binding.

Molecular cloning and functional expression of the pituitary adenylate cyclase-activating polypeptide type I receptor.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide belonging to the vasoactive intestinal peptide (VIP)/secretion/glucagon family of peptides, interacts with a distinct high-affinity receptor (type I receptor) on a number of tissues. These PACAP type I receptors have a high affinity for PACAP and a low affinity for VIP and are present in the hypothalamus and anterior pituitary, where they regulate the release of adrenocorticotropin, luteinizing hormone, growth hormone, and prolactin, and in the adrenal medulla, where they regulate the release of epinephrine. Type I PACAP receptors are also present in high concentrations in testicular germ cells, where they may regulate spermatogenesis, and some transformed cell lines, such as the rat pancreatic acinar carcinoma cell AR4-2J. Here we report the molecular cloning and functional expression of the PACAP type I receptor isolated from an AR4-2J cell cDNA library by cross-hybridization screening with a rat VIP receptor cDNA. The cDNA sequence encodes a unique 495-amino acid protein with seven transmembrane domains characteristic of guanine nucleotide-binding regulatory protein-coupled receptors. A high degree of sequence homology with the VIP, secretin, glucagon-like peptide 1, parathyroid, and calcitonin receptors suggests its membership in this subfamily of Gs-coupled receptors. Results of binding studies and stimulation of cellular cAMP accumulation in COS-7 cells transfected with this cDNA are characteristic of a PACAP type I receptor. Cloning of the PACAP type I receptor will enhance our understanding of its distribution, structure, and functional properties and ultimately increase our understanding of its physiological role.