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1.
Biomolecules ; 12(10)2022 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-36291631

RESUMO

Human melanocortin-2 receptor (hMC2R) co-expressed with the accessory protein mouse (m)MRAP1 in Chinese Hamster Ovary (CHO) cells has been used as a model system to investigate the activation and trafficking of hMC2R. A previous study had shown that the N-terminal domain of mMRAP1 makes contact with one of the extracellular domains of hMC2R to facilitate activation of hMC2R. A chimeric receptor paradigm was used in which the extracellular domains of hMC2R were replaced with the corresponding domains from Xenopus tropicalis MC1R, a receptor that does not interact with MRAP1, to reveal that EC2 (Extracellular domain 2) is the most likely contact site for hMC2R and mMRAP1 to facilitate activation of the receptor following an ACTH binding event. Prior to activation, mMRAP1 facilitates the trafficking of hMC2R from the ER to the plasma membrane. This process is dependent on the transmembrane domain (TM) of mMRAP1 making contact with one or more TMs of hMC2R. A single alanine substitution paradigm was used to identify residues in TM4 (i.e., I163, M165), EC2 (F167), and TM5 (F178) that play a role in the trafficking of hMC2R to the plasma membrane. These results provide further clarification of the activation mechanism for hMC2R.


Assuntos
Hormônio Adrenocorticotrópico , Receptor Tipo 2 de Melanocortina , Cricetinae , Humanos , Camundongos , Animais , Receptor Tipo 2 de Melanocortina/genética , Receptor Tipo 2 de Melanocortina/química , Receptor Tipo 2 de Melanocortina/metabolismo , Cricetulus , Células CHO , Hormônio Adrenocorticotrópico/metabolismo , Xenopus/metabolismo , Alanina
2.
Biosens Bioelectron ; 154: 112071, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32056965

RESUMO

In the neuroendocrine system, corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) play important roles in the regulation of the hypothalamic-pituitary-adrenal (HPA) system. Disorders of the HPA system lead to physiological problems, such as Addison's disease and Cushing's syndrome. Therefore, detection of CRH and ACTH is essential for diagnosing disorders related to the HPA system. Herein, receptors of the HPA axis were used to construct a bioelectronic sensor system for the detection of CRH and ACTH. The CRH receptor, corticotropin-releasing hormone receptor 1 (CRHR1), and the ACTH receptor, melanocortin 2 receptor (MC2R), were produced using an Escherichia coli expression system, and were reconstituted using nanodisc (ND) technology. The receptor-embedded NDs were immobilized on a floating electrode of a carbon nanotube field-effect transistor (CNT-FET). The constructed sensors sensitively detected CRH and ACTH to a concentration of 1 fM with high selectivity in real time. Furthermore, the reliable detection of CRH and ACTH in human plasma by the developed sensors demonstrated their potential in clinical and practical applications. These results indicate that CRHR1 and MC2R-based bioelectronic sensors can be applied for rapid and efficient detection of CRH and ACTH.


Assuntos
Hormônio Adrenocorticotrópico/isolamento & purificação , Técnicas Biossensoriais , Hormônio Liberador da Corticotropina/isolamento & purificação , Sistema Hipotálamo-Hipofisário/metabolismo , Doença de Addison/diagnóstico , Doença de Addison/genética , Hormônio Adrenocorticotrópico/química , Hormônio Liberador da Corticotropina/química , Síndrome de Cushing/diagnóstico , Síndrome de Cushing/genética , Humanos , Hidrocortisona/química , Hidrocortisona/genética , Sistema Hipófise-Suprarrenal/metabolismo , Receptor Tipo 2 de Melanocortina/química , Receptor Tipo 2 de Melanocortina/genética , Receptores da Corticotropina/química , Receptores da Corticotropina/genética , Receptores de Hormônio Liberador da Corticotropina/química , Receptores de Hormônio Liberador da Corticotropina/genética
3.
Int J Mol Sci ; 20(17)2019 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-31454910

RESUMO

The interaction between the pituitary hormone, adrenocorticotropin (ACTH), and melanocortin-2 receptor (MC2R) orthologs involves the H6 F7 R8 W9 and R/K15 K16 R17 R18 motifs in ACTH making contact with corresponding contact sites on MC2R. Earlier studies have localized the common HFRW binding site of all melanocortin receptors to residues in TM2, TM3, and TM6 that are located close to the extracellular space. The current study has identified residues in Xenopus tropicalis (xt) MC2R in TM4 (I158, F161), in EC2 (M166), and in TM5 (V172) that also are involved in activation of xtMC2R, and may be in the R/KKRR contact site of xtMC2R. These results are compared to earlier studies on the corresponding domains of human MC2R and rainbow trout MC2R in an effort to identify common features in the activation of teleost and tetrapod MC2R orthologs following stimulation with ACTH.


Assuntos
Receptor Tipo 2 de Melanocortina/metabolismo , Xenopus/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Sequência de Aminoácidos , Animais , Células CHO , Membrana Celular/metabolismo , Cricetulus , Humanos , Mutação , Receptor Tipo 2 de Melanocortina/agonistas , Receptor Tipo 2 de Melanocortina/química , Receptor Tipo 2 de Melanocortina/genética , Xenopus/genética
4.
Mol Cell Endocrinol ; 482: 11-17, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30553806

RESUMO

The adrenocorticotropic hormone (ACTH) receptor, known as the melanocortin-2 receptor (MC2R), plays a key role in regulating adrenocortical function. MC2R is a subtype of the melanocortin receptor family and ACTH is only agonist for MC2R. Our previous result indicates that ACTH1-17 is the minimal peptide required for MC2R activation but DPhe7-ACTH1-17 has no activity at MC2R. In this study, we examined the molecular basis of the MC2R responsible for ligand selectivity using ACTH analogues and MC2R mutagenesis. Our results indicate that substitution of the 3TM of the MC2R with the corresponding region of the MC3R switches DPhe-ACTH1-17 from no activity to agonist. Further experiment indicates that substitution of the amino acid residue leucine to isoleucine in 112 (L112I) of the 3TM of the MC2R changes both DPhe-ACTH1-17 and ACTH1-15 from no activity to agonists. Surprisingly, mutation L112I switches α-MSH from no activity to agonist, suggesting that this residue plays a key role at MC2R for ligand ACTH or α-MSH selectivity.


Assuntos
Hormônio Adrenocorticotrópico/metabolismo , Substituição de Aminoácidos , Receptor Tipo 2 de Melanocortina/química , Receptor Tipo 2 de Melanocortina/metabolismo , Hormônio Adrenocorticotrópico/análogos & derivados , Hormônio Adrenocorticotrópico/química , Sítios de Ligação , Células HEK293 , Humanos , Isoleucina/genética , Leucina/genética , Modelos Moleculares , Conformação Proteica , Receptor Tipo 2 de Melanocortina/genética , alfa-MSH/metabolismo
5.
Gen Comp Endocrinol ; 240: 182-190, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-27793723

RESUMO

The melanocortin receptor accessory proteins (MRAP and MRAP2) are small single-pass transmembrane proteins that regulate the biological functions of the melanocortin receptor (MCR) family. MCRs comprise five receptors (MC1R-MC5R) with diverse physiological roles in mammals. Five MCR members and two MRAPs were also predicted in the chicken (Gallus gallus) genome. However, little is known about their expression, regulation and biological functions. In this study, we cloned the MRAP and MRAP2 genes. Sequencing analysis revealed that the functional domains of MRAP and MRAP2 were conserved among species, suggesting that the physiological roles of chicken MRAP and MRAP2 could be similar to their mammalian counterparts. Tissue expression analysis demonstrated that MRAP was expressed in the adrenal gland, liver, spleen, glandular stomach and lungs, while MRAP2 is predominantly expressed in the adrenal gland. All five MCRs were present in the adrenal gland, but showed different expression patterns in other tissues. The MC5R was the only MCR member that was expressed in the chicken liver. The expression levels of MRAP in chicken liver were significantly increased at sexual maturity stage, and were significantly up-regulated (P<0.05) when chickens and chicken primary hepatocytes were treated with 17ß-estradiol in vivo and in vitro, respectively; however, expression levels of PPARγ were down-regulated, and no effect on MC5R was observed. Our results suggested that estrogen could stimulate the expression of MRAP in the liver of chicken through inhibiting the expression of transcription regulation factor PPARγ, and MRAP might play its biological role in a different way rather than forming an MRAP/MC2R complex in chicken liver during the egg-laying period.


Assuntos
Proteínas de Transporte/genética , Galinhas/genética , Estradiol/farmacologia , Fígado/metabolismo , Receptores de Melanocortina/genética , Sequência de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Clonagem Molecular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fígado/efeitos dos fármacos , PPAR gama/genética , PPAR gama/metabolismo , Filogenia , Receptor Tipo 2 de Melanocortina/química , Receptor Tipo 2 de Melanocortina/genética , Receptor Tipo 2 de Melanocortina/metabolismo , Receptores de Melanocortina/química , Receptores de Melanocortina/metabolismo , Alinhamento de Sequência , Distribuição Tecidual/efeitos dos fármacos
6.
J Biomol Struct Dyn ; 33(4): 770-88, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-24708442

RESUMO

Melanocortin system is composed of four peptide hormones namely α-, ß-, -γ, and adrenocorticotropic hormone (ACTH), derived from post-translational cleavage of a polypeptide precursor 'proopiomelanocortin (POMC).' Among these hormones, ACTH, a 38 amino acid residue peptide fragment is an important hormone as it is involved in steroid secretion. In addition to this, to cite a few, this hormone is also known to induce variety of other effects, such as alterations in motor/sexual behavior, improvement in memory, and anti-inflammatory effects. To date, five melanocortin receptors (MC1R-MC5R) have been characterized with tissue-specific expression patterns and different binding affinities for each of the melanocortin hormones to regulate various biological functions. In the present work, three-dimensional (3D) models of MC2R and ACTH from human have been predicted, followed by docking and molecular dynamics simulation. While the 3D model of MC2R receptor has been predicted through threading approach, structure of ACTH was built based on ab initio technique. The MC2R model was later successfully docked onto the ACTH structure. Molecular dynamics (MD) simulation for 20 ns was used to compute the binding free energy of MC2R with ACTH model under implicit solvent conditions.


Assuntos
Hormônio Adrenocorticotrópico/química , Receptor Tipo 2 de Melanocortina/química , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Termodinâmica
7.
Gen Comp Endocrinol ; 210: 145-51, 2015 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-24709361

RESUMO

Functional expression of the rainbow trout (rt) melanocortin-2 receptor (MC2R) in CHO cells requires co-expression with a teleost melanocortin-2 receptor accessory protein (MRAP) such as zebrafish (zf) MRAP. Transiently transfected rtMC2R/zfMRAP1 CHO cells were used to evaluate the efficacy of alanine substituted analogs of hACTH(1-24) in three motifs in the ligand: H(6)F(7)R(8)W(9), G(10)K(11)P(12)V(13)G(14), and K(15)K(16)R(17)R(18)P(19). Alanine substitution at all positions in each motif either completely blocked activation of the receptor (H(6)F(7)R(8)W(9) and K(15)K(16)R(17)R(18)P(19)) or resulted in just over 400 fold increase in EC50 value (G(10)K(11)P(12)V(13)G(14)). Single alanine substitutions in the H(6)F(7)R(8)W(9) motif indicated that substitution at either W(9) or R(8) resulted in a much larger increase in EC50 values as compared to substitutions at either F(7) or W(9). Alanine substitution at either K(15)K(16) or R(17)R(18)P(19) in the K(15)K(16)R(17)R(18)P(19) motif resulted in a statistically equivalent increase in EC50 value of at least 600 fold. Finally, alanine substitutions in the G(10)K(11)P(12)V(13)G(14) motif resulted in increases in EC50 values presumably as a result of altering the secondary structure of the ligand. However, truncated analogs of hACTH(1-24) in which either G(10)G(14) (ACTH(1-22), or K(11)P(12)V(13) (ACTH(1-21) were removed had no stimulatory activity. Finally, some of the hACTH(1-24) analogs were tested using rainbow trout head kidney pieces in vitro to confirm whether the response to analogs seen with the transient transfected rtMC2R CHO cells was similar to that of trout interrenal cells. The results of these alanine substitution analog studies are used to construct a multistep hypothetical model for the activation of teleost and tetrapod MC2Rs to account for the unique ligand selectivity of this receptor.


Assuntos
Oncorhynchus mykiss/metabolismo , Receptor Tipo 2 de Melanocortina , Hormônio Adrenocorticotrópico/química , Hormônio Adrenocorticotrópico/genética , Hormônio Adrenocorticotrópico/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Rim Cefálico , Ligantes , Oncorhynchus mykiss/genética , Estrutura Terciária de Proteína , Receptor Tipo 2 de Melanocortina/química , Receptor Tipo 2 de Melanocortina/genética , Receptor Tipo 2 de Melanocortina/metabolismo , Relação Estrutura-Atividade
8.
J Mol Endocrinol ; 53(2): 201-15, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25074265

RESUMO

The proteolysis of the pro-opiomelanocortin precursor results in the formation of melanocortins (MCs), a group of peptides that share the conserved -H-F-R-W- sequence, which acts as a pharmacophore for five subtypes of MC receptors (MCRs). MC type 2 receptor (MC2R; also known as ACTHR) is the most specialized of all the MCRs. It is predominantly expressed in the adrenal cortex and specifically binds ACTH. Unlike other MCRs, it requires melanocortin receptor accessory protein 1 (MRAP) for formation of active receptor and for its transport to the cell membrane. The molecular mechanisms underlying this specificity remain poorly understood. In this study, we used directed mutagenesis to investigate the role of various short MC2R sequence segments in receptor membrane trafficking and specific activation upon stimulation with ligands. The strategy of the study was to replace two to five amino acid residues within one MC2R segment with the corresponding residues of MC4R. In total, 20 recombinant receptors C-terminally fused to enhanced green fluorescent protein were generated and their membrane trafficking efficiencies and cAMP response upon stimulation with α-MSH and ACTH(1-24) were estimated during their stand-alone expression and coexpression with MRAP. Our results indicate that both the motif that determines the ligand-recognition specificity and the intracellular retention signal are formed by a specific extracellular structure, which is supported by the correct alignment of the transmembrane domains. Our results also indicate that the aromatic-residue-rich segment of the second extracellular loop is involved in the effects mediated by the second ACTH pharmacophore (-K-K-R-R-).


Assuntos
Domínios e Motivos de Interação entre Proteínas/genética , Receptor Tipo 2 de Melanocortina/genética , Receptor Tipo 2 de Melanocortina/metabolismo , Transdução de Sinais , Hormônio Adrenocorticotrópico/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular , AMP Cíclico/metabolismo , Expressão Gênica , Humanos , Dados de Sequência Molecular , Mutação , Receptor Tipo 2 de Melanocortina/química , Alinhamento de Sequência
9.
Mol Cell Endocrinol ; 394(1-2): 99-104, 2014 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-25017734

RESUMO

Cushing's disease, a hypercortisolemic state induced by an ACTH overexpressing pituitary adenoma, causes increased morbidity and mortality. Selective antagonism of the melanocortin type 2 receptor (MC2R) may be a novel treatment modality. Five structurally related peptides with modified HFRW sites but intact putative MC2R binding sites were tested for antagonistic activity at MC1R, MC2R/MRAP, MC3R, MC4R and MC5R. Two of these peptides (GPS1573 and GPS1574) dose-dependently antagonized ACTH-stimulated MC2R activity (IC50s of 66±23 nM and 260±1 nM, respectively). GPS1573 and 1574 suppressed the Rmax but not EC50 of ACTH on MC2R, indicating non-competitive antagonism. These peptides did not antagonize α-MSH stimulation of MC1R and antagonized MC3, 4 and 5R at markedly lower potency. GP1573 and GPS1574 antagonize MC4R with IC50s of 950 nM and 3.7 µM, respectively. In conclusion, two peptide antagonists were developed with selectivity for MC2R, forming a platform for development of a medical treatment for Cushing's disease.


Assuntos
Hormônio Adrenocorticotrópico/farmacologia , Desenho de Fármacos , Peptídeos/síntese química , Receptor Tipo 2 de Melanocortina/antagonistas & inibidores , Hormônio Adrenocorticotrópico/genética , Hormônio Adrenocorticotrópico/metabolismo , Sequência de Aminoácidos , Relação Dose-Resposta a Droga , Expressão Gênica , Células HEK293 , Humanos , Dados de Sequência Molecular , Peptídeos/farmacologia , Hipersecreção Hipofisária de ACTH/tratamento farmacológico , Ligação Proteica , Receptor Tipo 1 de Melanocortina/química , Receptor Tipo 1 de Melanocortina/genética , Receptor Tipo 1 de Melanocortina/metabolismo , Receptor Tipo 2 de Melanocortina/química , Receptor Tipo 2 de Melanocortina/genética , Receptor Tipo 2 de Melanocortina/metabolismo , Receptor Tipo 3 de Melanocortina/química , Receptor Tipo 3 de Melanocortina/genética , Receptor Tipo 3 de Melanocortina/metabolismo , Receptor Tipo 4 de Melanocortina/química , Receptor Tipo 4 de Melanocortina/genética , Receptor Tipo 4 de Melanocortina/metabolismo , Receptores de Melanocortina/química , Receptores de Melanocortina/genética , Receptores de Melanocortina/metabolismo , Relação Estrutura-Atividade , Transfecção
10.
Gen Comp Endocrinol ; 203: 3-9, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24713445

RESUMO

Following the biochemical characterization of the pituitary hormone, adrenocorticotropin (ACTH), in the 1950's, a number of structure/function studies were done which identifies two amino acid motifs in ACTH, the HFRW motif and KKRR motif, as critical for the activation of the "ACTH" receptor on adrenal cortex cells. In the 1990's the "ACTH" receptor was identified as a member of the melanocortin receptor gene family, and given the name melanocortin-2 receptor (MC2R). Since that time a number of studies on both tetrapod and teleost MC2R orthologs have established that these orthologs can only be activated by ACTH, but not by any of the MSH-sized melanocortin ligands, and these orthologs require interaction with the melanocortin-2 receptor accessory protein (MRAP) for functional expression. This review summarizes recent structure/function studies on human ACTH, and points out the importance of the GKPVG motif in ACTH for the activation of the receptor. In this regard, a multiple-step model for the activation of tetrapod and teleost MC2R orthologs is presented, and the evolution of gnathostome MC2R ligand selectivity and the requirement for MRAP interaction is discussed in light of a recent study on a cartilaginous fish MC2R ortholog. This review contains excerpts from the Gorbman/Bern Lecture presented at the Second Meeting of the North American Society for Comparative Endocrinology (NASCE).


Assuntos
Hormônio Adrenocorticotrópico/genética , Hormônio Adrenocorticotrópico/metabolismo , Evolução Molecular , Receptor Tipo 2 de Melanocortina/genética , Receptor Tipo 2 de Melanocortina/metabolismo , Hormônio Adrenocorticotrópico/química , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Receptor Tipo 2 de Melanocortina/química
11.
Gen Comp Endocrinol ; 205: 260-7, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-24726989

RESUMO

The chicken (Gallus gallus) melanocortin-2 receptor (cMC2R) can be functionally expressed in CHO cells when chicken melanocortin-2 receptor accessory protein 1 (cMRAP1) is co-expressed. The transiently transfected CHO cells responded in a robust manner to stimulation by hACTH(1-24) (EC50 value=2.7 × 10(-12)M +/- 1.3 × 10(-12)), but the transfected CHO cells could not be stimulated by NDP-MSH at concentrations as high as 10(-7)M. Incubation of cMC2R/cMRAP1 transfected cells with alanine substituted analogs of hACTH(1-24) at amino acid positions F(7) or W(9) completely blocked stimulation of the transfected cells. Similarly, incubation of cMC2R/cMRAP1 transfected cells with an analog of hACTH(1-24) with alanine substitutions at amino acid positions R(17)R(18)P(19) resulted in a 276 fold shift in EC50 value relative to the positive control (p<0.004). Collectively these observations suggest that cMC2R has binding sites for the HFRW motif and KKRRP motif of hACTH(1-24), and both motifs are required for full activation of the receptor. While previous studies had shown that Anolis carolinensis MC2R and Xenopus tropicalis MC2R could be functionally expressed in CHO cells that co-expressed mouse MRAP1, co-expression of these non-mammalian tetrapod MC2Rs with cMRAP1 resulted in a significant increase in sensitivity to hACTH(1-24), as measured by EC50 value, for A. carolinensis MC2R (p<0.005) and X. tropicalis MC2R (p<0.007). The implications of these observations are discussed.


Assuntos
Galinhas/metabolismo , Proteínas Modificadoras da Atividade de Receptores/farmacologia , Receptor Tipo 2 de Melanocortina/metabolismo , Hormônio Adrenocorticotrópico/metabolismo , Alanina , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Ligantes , Camundongos , Dados de Sequência Molecular , Proteínas Modificadoras da Atividade de Receptores/química , Proteínas Modificadoras da Atividade de Receptores/metabolismo , Receptor Tipo 2 de Melanocortina/química , Homologia de Sequência de Aminoácidos
12.
PLoS One ; 8(5): e65450, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23724142

RESUMO

The activation of melanocortin 2 receptor (MC2R) by ACTH mediates the signaling cascade leading to steroid synthesis in the interrenal tissue (analogous to the adrenal cortex in mammals) of fish. However, little is known about the functional regulation of this receptor in fish. In this work described, we cloned sea bass MC2R from a liver cDNA. SbMC2R requires the melanocortin 2 receptor accessory protein (MRAP) for its functional expression. Dietary cortisol but not long-term stress protocols downregulated interrenal sbMC2R expression. Data suggest the existence of a negative feedback on interrenal sbMC2R expression imposed by local or systemic glucocorticoids. This feedback could be involved in long-term stress adaptation by regulating interrenal sensitivity to ACTH. ACTH-induced MC2R activation stimulates hepatic lipolysis, suggesting that ACTH may mediate stress-induced effects upstream of cortisol release.


Assuntos
Adaptação Biológica/genética , Bass/genética , Bass/metabolismo , Receptor Tipo 2 de Melanocortina/genética , Receptor Tipo 2 de Melanocortina/metabolismo , Estresse Fisiológico/genética , Hormônio Adrenocorticotrópico/farmacologia , Sequência de Aminoácidos , Animais , Células CHO , Clonagem Molecular , Cricetulus , Jejum , Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Dados de Sequência Molecular , Especificidade de Órgãos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor Tipo 2 de Melanocortina/agonistas , Receptor Tipo 2 de Melanocortina/química , Alinhamento de Sequência
13.
Gen Comp Endocrinol ; 176(1): 9-17, 2012 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-22197208

RESUMO

Our previous studies showed that in barfin flounder, α-melanocyte-stimulating hormone (α-MSH) stimulates pigment dispersion in xanthophores, while it shows negligible effects in melanophores. The present study was undertaken to evaluate whether these results are limited to barfin flounder by using Japanese flounder. Three subtypes of proopiomelanocortin gene encoding melanocortins (MCs) were expressed in the Japanese flounder pituitary, one of which was also expressed in the skin. Expression of melanocortin 5 receptor gene (Mc5r) was observed in isolated xanthophores, while that of Mc1r and Mc5r was found in melanophores. In the xanthophores of Japanese flounder skin, α-MSH as well as desacetyl (Des-Ac)-α-MSH and diacetyl (Di-Ac)-α-MSH exhibited dose-dependent pigment-dispersing activities, indicating that the signals of α-MSH-related peptides were mediated by MC5R. On the other hand, α-MSH did not stimulate pigment dispersion in melanophores, while Des-Ac-α-MSH and Di-Ac-α-MSH did, thus indicating that the expression of two different types of Mcr is related to the decrease in α-MSH activity. Thus, the molecular repertoire in MC system observed in Japanese flounder is similar to that in barfin flounder. Moreover, the relationship between the pigment-dispersing activities of α-MSH-related peptides and the expression of Mcr subtypes in xanthophores and melanophores were also similar between Japanese flounder and barfin flounder. Consequently, we hypothesize that inhibition of α-MSH activity could be due to the formation of heterodimers comprising MC1R and MC5R, often observed in G-protein-coupled receptors.


Assuntos
Linguado/fisiologia , Melanóforos/fisiologia , Pigmentos Biológicos/fisiologia , alfa-MSH/fisiologia , Acetilação , Sequência de Aminoácidos , Animais , Dimerização , Dados de Sequência Molecular , Filogenia , Pró-Opiomelanocortina/genética , Receptor Tipo 1 de Melanocortina/química , Receptor Tipo 1 de Melanocortina/genética , Receptor Tipo 1 de Melanocortina/fisiologia , Receptor Tipo 2 de Melanocortina/química , Receptor Tipo 2 de Melanocortina/genética , Receptor Tipo 2 de Melanocortina/fisiologia , Receptor Tipo 4 de Melanocortina/química , Receptor Tipo 4 de Melanocortina/genética , Receptor Tipo 4 de Melanocortina/fisiologia , Receptores de Melanocortina/química , Receptores de Melanocortina/genética , Receptores de Melanocortina/fisiologia , Fenômenos Fisiológicos da Pele , Especificidade da Espécie
14.
Mol Endocrinol ; 25(11): 1961-77, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21920850

RESUMO

ACTH is the most important stimulus of the adrenal cortex. The precise molecular mechanisms underlying the ACTH response are not yet clarified. The functional ACTH receptor includes melanocortin-2 receptor (MC2R) and MC2R accessory proteins (MRAP). In human embryonic kidney 293/Flp recombinase target cells expressing MC2R, MRAP1 isoforms, and MRAP2, we found that ACTH induced a concentration-dependent and arrestin-, clathrin-, and dynamin-dependent MC2R/MRAP1 internalization, followed by intracellular colocalization with Rab (Ras-like small guanosine triphosphate enzyme)4-, Rab5-, and Rab11-positive recycling endosomes. Preincubation of cells with monensin and brefeldin A revealed that 28% of the internalized receptors were recycled back to the plasma membrane and participated in total accumulation of cAMP. Moreover, certain intracellular Ser and Thr (S/T) residues of MC2R were found to play important roles not only in plasma membrane targeting and function but also in promoting receptor internalization. The S/T residues T131, S140, T204, and S280 were involved in MRAP1-independent cell-surface MC2R expression. Other mutants (S140A, S208A, and S202D) had lower cell-surface expressions in absence of MRAPß. In addition, T143A and T147D drastically impaired cell-surface expression and function, whereas T131A, T131D, and S280D abrogated MC2R internalization. Thus, the modification of MC2R intracellular S/T residues may positively or negatively regulate its plasma membrane expression and the capacity of ACTH to induce cAMP accumulation. Mutations of T131, T143, T147, and S280 into either A or D had major repercussions on cell-surface expression, cAMP accumulation, and/or internalization parameters, pointing mostly to the second intracellular loop as being crucial for MC2R expression and functional regulation.


Assuntos
Receptor Tipo 2 de Melanocortina/metabolismo , Serina/química , Treonina/química , Arrestinas/metabolismo , Western Blotting , Linhagem Celular , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Dinaminas/metabolismo , Endossomos/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoprecipitação , Microscopia de Fluorescência , Ligação Proteica , Receptor Tipo 2 de Melanocortina/química , Receptor Tipo 2 de Melanocortina/genética , Serina/genética , Treonina/genética
15.
Eur J Pharmacol ; 660(1): 94-102, 2011 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-21211532

RESUMO

The melanocortin 2 (MC(2)) receptor differs from other melanocortin family members in its pharmacological profile and reliance on an accessory protein, MC(2) receptor accessory protein (MRAP), for surface expression and signal transduction. To identify features of the MC(2) receptor responsible for these characteristics, we created chimeras between MC(2) and MC(4) receptors and expressed these in CHO cells, where MRAP is essential for trafficking and signaling by MC(2) but not MC(4) receptors. Replacing the first transmembrane segment of the MC(2) receptor with the corresponding region from the MC(4) receptor allowed some surface expression in the absence of an accessory protein, while ACTH-induced cAMP production remained entirely MRAP-dependent. On the other hand, replacing the last two transmembrane domains, third extracellular loop and C-terminal tail of the MC(4) receptor with the corresponding regions from the MC(2) receptor resulted in MRAP-dependent signaling. Surprisingly, replacing the second and third transmembrane domains and the intervening first extracellular loop of MC(2) receptors with MC(4) sequences generated a chimera (2C2) that responded to both adrenocorticotropic hormone (ACTH) and to the potent MSH analog 4-norleucine-7-d-phenylalanine-α-melanocyte stimulating hormone (NDP-α-MSH), which does not activate native MC(2) receptors. The 2C2 chimeric receptor was able to respond to NDP-α-MSH without MRAP, but MRAP shifted the EC50 value for NDP-α-MSH to the left and caused constitutive activity. These results identify the first transmembrane domain as important for surface expression and regions from the second to third transmembrane segments of the MC(2) receptor as important for MRAP dependent-signal transduction and ligand specificity.


Assuntos
Proteínas de Membrana/metabolismo , Receptor Tipo 2 de Melanocortina/química , Receptor Tipo 2 de Melanocortina/metabolismo , Receptor Tipo 4 de Melanocortina/química , Receptor Tipo 4 de Melanocortina/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Humanos , Ligantes , Camundongos , Transporte Proteico , Receptor Tipo 2 de Melanocortina/genética , Receptor Tipo 4 de Melanocortina/genética , Proteínas Recombinantes de Fusão/genética , Transdução de Sinais , Especificidade por Substrato
16.
Mol Cell Endocrinol ; 321(2): 175-83, 2010 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-20206229

RESUMO

The adrenocorticotropic hormone (ACTH) receptor has highly specific membrane expression that is limited to adrenal cells; in other cell types the polypeptide fails to be transported to the cell surface. Unlike other evolutionarily related members of the melanocortin receptor family (MC1R-MC5R) that recognize different melanocortin peptides, ACTHR (MC2R) binds only ACTH. We used a mutagenesis approach involving systematic construction of chimeric ACTHR/MC4R receptors to identify the domains determining the selectivity of ACTHR membrane transport and ACTH binding. In total 15 chimeric receptors were created by replacement of selected domains of human ACTHR with the corresponding regions of human MC4R. We developed an analytical method to accurately quantify cell-membrane localization of recombinant receptors fused with enhanced green fluorescent protein by confocal fluorescence microscopy. The chimeric receptors were also tested for their ability to bind ACTH (1-24) and the melanocyte-stimulating hormone (MSH) analog, Nle4, DPhe7-alpha-MSH, and to induce a cAMP response. Our results indicate that substitution of the MC4R N-terminal segment with the homologous segment of ACTHR significantly decreased membrane transport. We also identified another signal localized in the third and fourth transmembrane regions as the main determinant of ACTHR intracellular retention. In addition, we found that the fourth and fifth transmembrane domains of the ACTHR are involved in ACTH binding selectivity. We discuss the mechanisms involved in bypassing these arrest signals via an interaction with melanocortin 2 receptor accessory protein (MRAP) and the possible mechanisms that determine the high ligand-binding specificity of ACTHR.


Assuntos
Ligantes , Transporte Proteico/fisiologia , Receptor Tipo 2 de Melanocortina/química , Receptor Tipo 2 de Melanocortina/metabolismo , Linhagem Celular , Corantes Fluorescentes/química , Humanos , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Microscopia Confocal , Estrutura Terciária de Proteína , Especificidade por Substrato
18.
J Clin Endocrinol Metab ; 93(8): 3097-105, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18492762

RESUMO

CONTEXT: Familial glucocorticoid deficiency (FGD) is an autosomal recessive disorder characterized by unresponsiveness to ACTH. In this study, two mutations of the ACTH receptor (MC2R) gene are reported in this FGD clinical case. OBJECTIVE: The objective of the study was to characterize a novel MC2R gene mutation in a compound heterozygous patient with FGD phenotype. DESIGN: This was a clinical case description, biochemical, molecular, and bioinformatics analysis to describe a novel MC2R gene mutation. PATIENTS: The subject of the study was a male diagnosed with primary adrenal insufficiency. The family history showed nonconsanguineous healthy parents, three healthy siblings, and one brother affected with FGD. MAIN OUTCOME MEASURES: The mutant MC2R-Ala126Ser showed significantly lower activity when it was stimulated with ACTH-(1-24) than did cells transfected with wild-type MC2R. RESULTS: The molecular studies demonstrated the presence of an adenine heterozygous insertion (InsA1347) in the MC2R gene (G217fs) in the patient. This insertion was due to a frame shift mutation in one allele and a premature stop codon codifying an aberrant receptor of 247 residues (27.2 kDa). We also found a novel heterozygous mutation alanine 126 by serine. Molecular dynamic simulations showed that serine 126 side chain fluctuates forming a noncanonical intrahelical hydrogen bond in the transmembrane helix 3 of the mutated receptor. This produces a structural rearrangement of the MC2R internal cavities that may affect the ligand recognition and signal transduction throughout the G protein. CONCLUSIONS: We propose a molecular explanation for the reduced activity exhibited by the MC2R alanine 126 by serine mutant.


Assuntos
Insuficiência Adrenal/genética , Glucocorticoides/deficiência , Receptor Tipo 2 de Melanocortina/genética , Animais , Células CHO , Pré-Escolar , Cricetinae , Cricetulus , Humanos , Masculino , Modelos Moleculares , Mutação , Receptor Tipo 2 de Melanocortina/química , Receptor Tipo 2 de Melanocortina/fisiologia
19.
Biochemistry ; 46(40): 11389-97, 2007 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-17877367

RESUMO

The melanocortin-2 receptor (MC2R), also known as the adrenocorticotropic hormone (ACTH) receptor, plays an important role in regulating and maintaining adrenocortical function, specifically steroidogenesis. Mutations of the human MC2R (hMC2R) gene have also been identified in humans with familial glucocorticoid deficiency; however, the molecular basis responsible for hMC2R ligand binding and signaling remains unclear. In this study, both truncated ACTH peptides and site-directed mutagenesis studies were used to determine molecular mechanisms of hMC2R binding ACTH and signaling. Our results indicate that ACTH1-16 is the minimal peptide required for hMC2R binding and signaling. Mutations of common melanocortin receptor family amino acid residues E80 in transmembrane domain 2 (TM2), D107 in TM3, F178 in TM4, F235 and H238 in TM6, and F258 in TM7 significantly reduced ACTH-binding affinity and signaling. Furthermore, mutations of unique amino acids D104 and F108 in TM3 and F168 and F178 in TM4 significantly decreased ACTH binding and signaling. In conclusion, our results suggest that the residues in TM2, TM3, and TM6 of hMC2R share similar binding sites with other MCRs but the residues identified in TM4 and TM7 of hMC2R are unique and required for ACTH selectivity. Our study suggests that hMC2R may have a broad binding pocket in which both conserved and unique amino acid residues are required, which may be the reason why alpha-MSH was not able to bind hMC2R.


Assuntos
Hormônio Adrenocorticotrópico/metabolismo , Receptor Tipo 2 de Melanocortina/metabolismo , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , AMP Cíclico/metabolismo , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Receptor Tipo 2 de Melanocortina/química , Receptor Tipo 2 de Melanocortina/genética , Transdução de Sinais/genética , Transfecção
20.
Am J Physiol Regul Integr Comp Physiol ; 293(3): R1120-6, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17596328

RESUMO

The ACTH receptor, also known as the melanocortin-2 receptor (MC2R), is critical for ACTH-mediated adrenal glucocorticoid release. Human MC2R (hMC2R) has 10 cysteine residues, which are located in extracellular loops (ELs), transmembrane domains (TMs), and intracellular loops (ILs). In this study, we examined the importance of these cysteine residues in receptor function and determined their involvement in disulfide bond formation. We replaced these cysteines with serine and expressed the mutated receptors in adrenal OS3 cells, which lack endogenous MC2R. Our results indicate that four mutations, C21S in NH(2) terminus, C245S, C251S, and C253S in EL3, resulted in significant decrease both in receptor expression and receptor function. Mutation of cysteine 231 in TM6 significantly decreased ACTH binding affinity and potency. In contrast, the five other mutated receptors (C64S, C158S, C191S, C267S, and C293S) did not significantly alter ACTH binding affinity and potency. These results suggest that extracellular cysteine residue 21, 245, 251, and 253, as well as transmembrane cysteine residue 231 are crucial for ACTH binding and signaling. Further experiments suggest that a disulfide bond exists between the residue C245 and C251 in EL3. These findings provide important insights into the importance of cysteine residues of hMC2R for receptor function.


Assuntos
Cisteína/química , Receptor Tipo 2 de Melanocortina/química , Receptor Tipo 2 de Melanocortina/fisiologia , Hormônio Adrenocorticotrópico/metabolismo , Sequência de Aminoácidos , Linhagem Celular , AMP Cíclico/metabolismo , Dissulfetos/metabolismo , Ditiotreitol/farmacologia , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Reagentes de Sulfidrila/farmacologia , Transfecção
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