Failed export of the adrenocorticotrophin receptor from the endoplasmic reticulum in non-adrenal cells: evidence in support of a requirement for a specific adrenal accessory factor

Noon, Luke A.; Franklin, Jade M.; King, Peter; Goulding, N J; Hunyady, László; Clark, Adrian J. L.

doi:10.1677/joe.0.1740017

Cited by 81 publications

(46 citation statements)

References 44 publications

(44 reference statements)

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“…Clonal selection of H295R cells, specifically with focus on clones with increased ACTH response, has produced HAC15 cells, but these cells do not have the responsiveness seen in primary cultures (Parmar et al 2008, Xing et al 2010, 2011. The existence of an MC2R accessory factor had been suspected because heterologous expression of MC2R does not yield a functional receptor in most cell types (Noon et al 2002). In 2005, MRAP was identified to cause FGD type 2 (Metherell et al 2005).…”

Section: Disscusionmentioning

confidence: 99%

H295R expression of melanocortin 2 receptor accessory protein results in ACTH responsiveness

Nanba¹,

Chen²,

Turcu³

et al. 2015

Journal of Molecular Endocrinology

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The H295R adrenocortical cell line is widely used for molecular analysis of adrenal functions but is known to have only modest ACTH responsiveness. The lack of ACTH response was linked to a low expression of its receptor, melanocortin 2 receptor (MC2R). We hypothesized that increasing the MC2R accessory protein (MRAP), which is required to traffic MC2R from the endoplasmic reticulum to the cell surface, would increase ACTH responsiveness. Lentiviral particles containing human MRAP-open reading frame were generated and transduced in H295R cells. Using antibiotic resistance, 18 clones were isolated for characterization. The most ACTH-responsive steroidogenic clone, H295RA, was used for further experiments. Successful induction of MRAP and increased expression of MC2R in H295RA cells was confirmed by quantitative real-time RT-PCR and protein analysis. Treatment with ACTH significantly increased aldosterone, cortisol, and dehydroepiandrosterone production in H295RA cells. ACTH also significantly increased transcript levels for all of the steroidogenic enzymes required to produce aldosterone, cortisol, and dehydroepiandrosterone, as well as MC2R mRNA. Using liquid chromatography/tandem mass spectrometry, we further revealed that the main unconjugated steroids produced in H295RA cells were 11-deoxycortisol, cortisol, and androstenedione. Treatment of H295RA cells with ACTH also acutely increased cAMP production and cellular protein levels for total and phosphorylated steroidogenic acute regulatory protein. In summary, through genetic manipulation, we have developed an ACTH-responsive human adrenocortical cell line. The cell line will provide a powerful in vitro tool for molecular analysis of physiologic and pathologic conditions involving the hypothalamic-pituitary-adrenal axis.

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Section: Disscusionmentioning

confidence: 99%

H295R expression of melanocortin 2 receptor accessory protein results in ACTH responsiveness

Nanba¹,

Chen²,

Turcu³

et al. 2015

Journal of Molecular Endocrinology

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“…This result may be explained by low expression of the MC2R in A and R cells. Alternatively, studies by others suggested that poor stimulation by ACTH may be due to impaired trafficking of the receptor from the endoplasmic reticulum to the cell surface (Noon et al 2002).…”

Section: Expression Of At1 and Mc2r And The Acth/camp Signalingmentioning

confidence: 99%

Human adrenal corticocarcinoma NCI-H295R cells produce more androgens than NCI-H295A cells and differ in 3β-hydroxysteroid dehydrogenase type 2 and 17,20 lyase activities

Samandari¹,

Kempná²,

Nuoffer³

et al. 2007

Journal of Endocrinology

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The human adrenal cortex produces mineralocorticoids, glucocorticoids, and androgens in a species-specific, hormonally regulated, zone-specific, and developmentally characteristic fashion. Most molecular studies of adrenal steroidogenesis use human adrenocortical NCI-H295A and NCI-H295R cells as a model because appropriate animal models do not exist. NCI-H295A and NCI-H295R cells originate from the same adrenocortical carcinoma which produced predominantly androgens but also smaller amounts of mineralocorticoids and glucocorticoids. Research data obtained from either NCI-H295A or NCI-H295R cells are generally compared, although for the same experiments no direct comparison between the two cell lines has been performed. Therefore, we compared the steroid profile and the expression pattern of important genes involved in steroidogenesis in both cell lines. We found that steroidogenesis differs profoundly. NCI-H295A cells produce more mineralocorticoids, whereas NCI-H295R cells produce more androgens. Expression of the 3b-hydroxysteroid dehydrogenase (HSD3B2), cytochrome b5, and sulfonyltransferase genes is higher in NCI-H295A cells, whereas expression of the cytochrome P450c17 (CYP17), 21-hydroxylase (CYP21), and P450 oxidoreductase genes does not differ between the cell lines. We found lower 3b-hydroxysteroid dehydrogenase type 2 but higher 17,20-lyase activity in NCI-H295R cells explaining the 'androgenic' steroid profile for these cells and resembling the zona reticularis of the human adrenal cortex. Both cell lines were found to express the ACTH receptor at low levels consistent with low stimulation by ACTH. By contrast, both cell lines were readily stimulated by 8Br-cAMP. The angiotensin type 1 receptor was highly expressed in NCI-H295R than NCI-H295A cells and angiotensin II stimulated steroidogenesis in NCI-H295R but not NCI-H295A cells. Our data suggest that comparative studies between NCI-H295A and NCI-H295R cells may help find important regulators of mineralocorticoid or androgen biosynthesis.

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“…In their original paper, Mountjoy and coworkers (Mountjoy et al 1992) only reported MC2R expression in a cell line that expressed an endogenous MC1R. Using a green fluorescent protein-tagged MC2R, we demonstrated that the hybrid protein seemed to be retained in the endoplasmic reticulum and failed to reach the cell surface (Noon et al 2002). Significantly, cell lines derived from the murine adrenocortical tumor Y1 line, which had developed unresponsiveness to ACTH action were found to be capable of expressing the transfected MC2R (Yang et al 1997).…”

Section: The Discovery Of Mrapmentioning

confidence: 78%

Promiscuity among the MRAPs

Clark

Chan

2017

Journal of Molecular Endocrinology

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The melanocortin 2 receptor accessory protein (MRAP) was originally discovered to be an essential co-receptor for the ACTH receptor/melanocortin 2 receptor, and it physically interacts with this receptor and is required for receptor trafficking and ligand binding. A related molecule, MRAP2, is mainly expressed in the CNS and appears to have a role with the melanocortin 4 receptor. Consistent with this is the observation that a massively obese phenotype develops when the Mrap2 gene is deleted in mice. However, the characteristics of this phenotype differ from those of Mc4r-deleted mice and suggest that an additional role, possibly resulting from an interaction with other receptors is possible. In support of this, a functional interaction with the prokineticin receptors was recently reported. Evidence for other receptor interactions and aspects of the tissue distribution of MRAP and MRAP2 gene expression may indicate that these accessory proteins have a wider role than with the melanocortin receptors alone.

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Failed export of the adrenocorticotrophin receptor from the endoplasmic reticulum in non-adrenal cells: evidence in support of a requirement for a specific adrenal accessory factor

Cited by 81 publications

References 44 publications

H295R expression of melanocortin 2 receptor accessory protein results in ACTH responsiveness

H295R expression of melanocortin 2 receptor accessory protein results in ACTH responsiveness

Human adrenal corticocarcinoma NCI-H295R cells produce more androgens than NCI-H295A cells and differ in 3β-hydroxysteroid dehydrogenase type 2 and 17,20 lyase activities

Promiscuity among the MRAPs

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