[7] Functional assays to study coupling of dopamine D2 receptors to G proteins

Baik, Ja Hyun; Bhatia, Madhav; Picetti, Roberto; Thiriet, G.; Borrelli, Emiliana

doi:10.1016/s1043-9471(96)80043-7

Cited by 2 publications

(7 citation statements)

References 19 publications

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“…Final PCR products of each chimera were isolated and purified from agarose gel. All chimeric inserts were subsequently subcloned into pSV (16,25) using the BamHI and XhoI enzyme restriction sites. All chimeric inserts were sequenced on an automatic sequencer, ALFexpress (Amersham Biosciences), using a dideoxy terminator cycle sequencing kit (Amersham Biosciences).…”

Section: Methodsmentioning

confidence: 99%

“…Expression of Melanocortin Receptors and Luciferase Reporter Gene Assay-Rat MC3R and human MC4R cDNA, kindly provided by Dr. Roger D. Cone, were cloned into pSV expression vector (16,25). For receptor expression, HEK 293T cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and transfected with pSV-rMC3R and pSV-hMC4R, respectively, using FuGENE 6 transfection reagent (Roche Molecular Biochemicals) using the procedure recommended by the manufacturer.…”

Section: Methodsmentioning

confidence: 99%

“…Therefore, it is suggested that this differential inositol phospholipid signaling by MC3R and MC4R may also contribute to the differential CRE-luciferase activity mediated by these two receptors. To a certain extent, because a CRE-reporter gene assay can effectively monitor both G s and G q activation by CREB-mediated gene expression (24,25), this distinct inositol phospholipid signaling would be one of the possible explanations for the differential CRE-reporter gene activation profile between MC3R and MC4R. On the other hand, the basal level of the reporter gene activity with MC4R in the absence of stimulation with the melanocortin analogue is actually higher than that of MC3R, suggesting that MC4R could be constitutively activated.…”

Section: Fig 4-continuedmentioning

confidence: 99%

“…We used the CRE-luciferase reporter gene assay to score the efficacy of receptor-G proteins coupling (24,25). In parallel, amino acid mutations were generated in the third intracytoplasmic loop of MC4R to identify the residues that play a role in G-protein coupling.…”

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confidence: 99%

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Identification of Domains Directing Specificity of Coupling to G-proteins for the Melanocortin MC3 and MC4 Receptors

Kim

Lee

Kim

et al. 2002

Journal of Biological Chemistry

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The melanocortin receptors, MC3R and MC4R, are G protein-coupled receptors that are involved in regulating energy homeostasis. Using a luciferase reporter gene under the transcriptional control of a cAMPresponsive element (CRE), the coupling efficiency of the MC4R and MC3R to G-proteins was previously shown to be different. MC4R exhibited only 30 -50% of the maximum activity induced by MC3R. To assess the role of the different MC3R and MC4R domains in G-protein coupling, several chimeric MC3R/MC4R receptors were constructed. The relative luciferase activities, which were assessed after transfecting the chimeric receptors into HEK 293T cells, showed that the i3 (3rd intracellular) loop domain has an essential role in the differential signaling of MC3R and MC4R. To reveal which amino acid residue was involved in the MC4R-specific signaling in the i3 loop, a series of mutant MC4Rs was constructed. Reporter gene analysis showed that single mutations of Arg 220 to Ala and Thr 232 to either Val or Ala increased the relative luciferase activities, which suggests that these specific amino acids, Arg 220 and Thr 232 , in the i3 loop of MC4R play crucial roles in G-protein coupling and the subtype-specific signaling pathways. An examination of the inositol phosphate (IP) levels in the cells transfected with either MC3R or MC4R after being exposed to the melanocortin peptides revealed significant stimulation of IP production by MC3R but no detectable increase in IP production was observed by MC4R. Furthermore, none of the MC4R mutants displayed melanocortin peptide-stimulated IP production. Overall, this study demonstrated that MC3R and MC4R have distinct signaling in either the cAMP-or the inositol phospholipid-mediated pathway with different conformational requirements.Melanocortins are peptide hormones that are derived from the precursor peptide pro-opiomelanocortin, by a series of proteolytic cleavages (1). The melanocortins are known to have a broad spectrum of physiological actions, which include the regulation of melanocyte pigmentation (2), thermoregulation (3), obesity (4), control of the cardiovascular system (5), and learning and memory (6), and have also been found to have immunomodulatory effects (7). These hormones mediate their effects through G protein-coupled receptors by stimulating adenylate cyclase (8). To date five melanocortin receptor subtypes, with different patterns of tissue expression in the brain and peripheral body, have been cloned and characterized (8 -12).It has been reported that the activation of melanocortin 4 receptor (MC4R) 1 by ␣-melanocyte-stimulating hormone (MSH) increases energy expenditure and decreases food intake. Moreover, the genetic disruption of MC4R was found to cause obesity in mice (13). Recent experiments in MC3R-null mice indicate that the inactivation of MC3R results in increased fat mass and reduced body mass, despite the fact that the animals were hypophagic and maintained normal metabolic rates (14, 15). These results suggest the nonredundancy of the MC3R and MC4R m...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Fig 4-continuedmentioning

confidence: 99%

mentioning

confidence: 99%

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Identification of Domains Directing Specificity of Coupling to G-proteins for the Melanocortin MC3 and MC4 Receptors

Kim

Lee

Kim

et al. 2002

Journal of Biological Chemistry

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“…Elevation of intracellular cAMP results in phosphorylation of the transcription factor CREB by protein kinase A, which in turn activates the transcription factor. This system has a number of advantages that allow the detection of subtle but physiologically relevant changes in intracellular cAMP levels as represented by changes in cAMP‐mediated gene expression [16–18]. Hence, this assay is likely to provide evidence for the receptor–G‐protein–effector interaction.…”

mentioning

confidence: 99%

Differential regulation of cAMP‐mediated gene transcription and ligand selectivity by MC3R and MC4R melanocortin receptors

Lee¹,

Lee²,

Jung³

et al. 2001

European Journal of Biochemistry

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Melanocortins are known to be involved in the regulation of feeding behavior. These hormones mediate their effects through G-protein-coupled receptors by stimulating adenylate cyclase. In this study we describe the functional response of melanocortin 4 receptor (MC4R) and melanocortin 3 receptor (MC3R) in HEK 293T cells, by using a luciferase reporter gene under the transcriptional control of a cAMP-responsive element (CRE) as a monitor of intracellular cAMP levels and cAMP-regulated gene expression. We were able to show that MC4R and MC3R expressed in the human cell line HEK 293T stimulate transcription induced by stimulation with different analogs of a-melanocyte-stimulating hormone (a-MSH) at different levels. In our assay of CRE-mediated gene transcription activity, a-MSH-ND was the most efficient a-MSH analog for MC4R whereas NDP-MSH was the most efficient for MC3R. Changing the His6 residue of a-MSH-ND to Gln or Lys markedly decreased CRE-mediated luciferase activity for MC3R compared with MC4R. On analysis by modeling the receptor±ligand complex by NMR, [Gln6]a-MSH-ND and [Lys6]a-MSH-ND showed different conformational interactions between MC3R and MC4R. Furthermore, the maximum coupling efficiency of MC4R and MC3R to G proteins was different; MC4R showed only 30±50% of the maximum activity induced by MC3R. In total, our results suggest that a differential receptor±ligand interaction is involved and that the relative interactions of MC3R and MC4R with G protein are possibly quantitatively and qualitatively different.Keywords: cAMP; G protein; melanocortin; molecular modeling; obesity.Melanocortins are peptide hormones derived through a series of proteolytic cleavages from the precursor peptide pro-opiomelanocortin [1]. They are known to have a broad spectrum of physiological actions including regulation of melanocyte pigmentation [2], thermoregulation [3], regulation of obesity [4], control of the cardiovascular system [5] and learning and memory [6], as well as immunomodulatory effects [7]. These hormones mediate their effects through G-protein-coupled receptors by stimulating adenylate cyclase [8]. To date, five melanocortin receptor subtypes with different patterns of tissue expression in brain and the peripheral body have been cloned and characterized [8±12].It has been reported that activation of melanocortin 4 receptor (MC4R) by a-melanocyte-stimulating hormone (a-MSH) increases energy expenditure and decreases food intake; genetic disruption of MC4R also caused obesity in mice [13]. Thus, a-MSH derived from the arcuate nucleus pro-opiomelanocortin neurons, the primary source of ligands for MC4R, appears to play an inhibitory role in feeding and energy storage. Therefore, MC4R-selective agonists have been considered as potential candidates for the treatment of abnormal eating behavior including obesity and anorexia. MC3R is also abundantly expressed in the brain and peripheral tissues such as the placenta, gut tissues and the human heart [14]. Recent experiments in MC3R-null mice indicate that inacti...

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[7] Functional assays to study coupling of dopamine D2 receptors to G proteins

Cited by 2 publications

References 19 publications

Identification of Domains Directing Specificity of Coupling to G-proteins for the Melanocortin MC3 and MC4 Receptors

Identification of Domains Directing Specificity of Coupling to G-proteins for the Melanocortin MC3 and MC4 Receptors

Differential regulation of cAMP‐mediated gene transcription and ligand selectivity by MC3R and MC4R melanocortin receptors

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