Identification of domains directing specificity of coupling to G-proteins for the melanocortin MC3 and MC4 receptors

Chung Sub Kim, Soo Hyun Lee, Ryang Yeo Kim, Byung Jin Kim, Song Zhe Li, In Hye Lee, Eun Jin Lee, Sungkil Lim, Yun Soo Bae, Weon Tae Lee, Ja Hyun Baik

Research output: Contribution to journalArticle

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Abstract

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 cAMP-responsive 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.

Original languageEnglish
Pages (from-to)31310-31317
Number of pages8
JournalJournal of Biological Chemistry
Volume277
Issue number35
DOIs
Publication statusPublished - 2002 Aug 30

Fingerprint

Receptor, Melanocortin, Type 3
Melanocortin Receptors
Receptor, Melanocortin, Type 4
Melanocortins
Inositol Phosphates
GTP-Binding Proteins
Luciferases
Reporter Genes
Genes
Amino Acids
Peptides
HEK293 Cells
G-Protein-Coupled Receptors
Phosphatidylinositols
Homeostasis
Mutation

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Kim, Chung Sub ; Lee, Soo Hyun ; Kim, Ryang Yeo ; Kim, Byung Jin ; Li, Song Zhe ; Lee, In Hye ; Lee, Eun Jin ; Lim, Sungkil ; Bae, Yun Soo ; Lee, Weon Tae ; Baik, Ja Hyun. / Identification of domains directing specificity of coupling to G-proteins for the melanocortin MC3 and MC4 receptors. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 35. pp. 31310-31317.
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title = "Identification of domains directing specificity of coupling to G-proteins for the melanocortin MC3 and MC4 receptors",
abstract = "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 cAMP-responsive 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.",
author = "Kim, {Chung Sub} and Lee, {Soo Hyun} and Kim, {Ryang Yeo} and Kim, {Byung Jin} and Li, {Song Zhe} and Lee, {In Hye} and Lee, {Eun Jin} and Sungkil Lim and Bae, {Yun Soo} and Lee, {Weon Tae} and Baik, {Ja Hyun}",
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Identification of domains directing specificity of coupling to G-proteins for the melanocortin MC3 and MC4 receptors. / Kim, Chung Sub; Lee, Soo Hyun; Kim, Ryang Yeo; Kim, Byung Jin; Li, Song Zhe; Lee, In Hye; Lee, Eun Jin; Lim, Sungkil; Bae, Yun Soo; Lee, Weon Tae; Baik, Ja Hyun.

In: Journal of Biological Chemistry, Vol. 277, No. 35, 30.08.2002, p. 31310-31317.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Identification of domains directing specificity of coupling to G-proteins for the melanocortin MC3 and MC4 receptors

AU - Kim, Chung Sub

AU - Lee, Soo Hyun

AU - Kim, Ryang Yeo

AU - Kim, Byung Jin

AU - Li, Song Zhe

AU - Lee, In Hye

AU - Lee, Eun Jin

AU - Lim, Sungkil

AU - Bae, Yun Soo

AU - Lee, Weon Tae

AU - Baik, Ja Hyun

PY - 2002/8/30

Y1 - 2002/8/30

N2 - 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 cAMP-responsive 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.

AB - 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 cAMP-responsive 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.

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