Clinical, Genetic, and Functional Characterization of Adrenocorticotropin Receptor Mutations Using a Novel Receptor Assay

Department of Pediatrics and Metabolic Research Unit, University of California, San Francisco, California 94143-0978, USA.
Journal of Clinical Endocrinology &amp Metabolism (Impact Factor: 6.21). 10/2002; 87(9):4318-23. DOI: 10.1210/jc.2002-020501
Source: PubMed


The ACTH receptor (MC2R) is expressed predominantly in the adrenal cortex, but is one of five G protein-coupled, seven-transmembrane melanocortin receptors (MCRs), all of which bind ACTH to some degree. Testing of MC2R activity is difficult because most cells express endogenous MCRs; hence, ACTH will elicit background activation of assayable reporter systems. Inactivating mutations of MC2R lead to hereditary unresponsiveness to ACTH, also known as familial glucocorticoid deficiency (FGD). These patients are usually seen in early childhood with very low cortisol concentrations, normal mineralocorticoids, hyperpigmentation, and increased bodily growth. Several MC2R mutations have been reported in FGD, but assays of the activities of these mutants are cumbersome. We saw two patients with typical clinical findings of FGD. Genetic analysis showed that patient 1 was homozygous for the mutation R137W, and patient 2 was a compound heterozygote for S74I and Y254C. We tested the activity of these mutations in OS-3 cells, which are unresponsive to ACTH but have intact downstream cAMP signal transduction. OS-3 cells transfected with a cAMP-responsive luciferase reporter plasmid (pCREluc) were unresponsive to ACTH, but cotransfection with a vector expressing human MC2R increased luciferase activity more than 40-fold. Addition of ACTH to cells cotransfected with the pCREluc reporter and wild-type MC2R activated luciferase expression with a 50% effective concentration of 5.5 x 10(-9) M ACTH, which is similar to previously reported values. By contrast, the MC2R mutant R137W had low activity, and the S74I or Y254C mutants elicited no measurable response. This assay provides excellent sensitivity in an easily assayed transient transfection system, providing a more rapid and efficient measurement of ACTH receptor activity.

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    • "When stable cell lines were used (WT-V2R or R137L-V2R), cells were transfected only with the CRE-luciferase reporter plasmid. Twentyfour hours after transfection, cells were lysed and assayed for luciferase activity by using the Dual Luciferase Reporter Assay System (Promega) as described previously (Stables et al., 1999; Flü ck et al., 2002). Data are expressed as the mean luciferase activity, in arbitrary units, and normalized to WT-V2R-promoted luciferase activity. "
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    ABSTRACT: Substitution of arginine-137 of the vasopressin type 2 receptor (V2R) for histidine (R137H-V2R) leads to nephrogenic diabetes insipidus (NDI), whereas substitution of the same residue to cysteine or leucine (R137C/L-V2R) causes the nephrogenic syndrome of inappropriate antidiuresis (NSIAD). These two diseases have opposite clinical outcomes. Still, the three mutant receptors were shown to share constitutive beta-arrestin recruitment and endocytosis, resistance to vasopressin-stimulated cAMP production and mitogen-activated protein kinase activation, and compromised cell surface targeting, raising questions about the contribution of these phenomenons to the diseases and their potential treatments. Blocking endocytosis exacerbated the elevated basal cAMP levels promoted by R137C/L-V2R but not the cAMP production elicited by R137H-V2R, demonstrating that substitution of Arg137 to Cys/Leu, but not His, leads to constitutive V2R-stimulated cAMP accumulation that most likely underlies NSIAD. The constitutively elevated endocytosis of R137C/L-V2R attenuates the signaling and most likely reduces the severity of NSIAD, whereas the elevated endocytosis of R137H-V2R probably contributes to NDI. The constitutive signaling of R137C/L-V2R was not inhibited by treatment with the V2R inverse agonist satavaptan (SR121463). In contrast, owing to its pharmacological chaperone property, SR121463 increased the R137C/L-V2R maturation and cell surface targeting, leading to a further increase in basal cAMP production, thus disqualifying it as a potential treatment for patients with R137C/L-V2R-induced NSIAD. However, vasopressin was found to promote beta-arrestin/AP-2-dependent internalization of R137H/C/L-V2R beyond their already elevated endocytosis levels, raising the possibility that vasopressin could have a therapeutic value for patients with R137C/L-V2R-induced NSIAD by reducing steady-state surface receptor levels, thus lowering basal cAMP production.
    Molecular pharmacology 02/2010; 77(5):836-45. DOI:10.1124/mol.109.061804 · 4.13 Impact Factor
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    • "All five of these receptors can bind ACTH to some extent, but MC2R binds ACTH at the highest affinity, is expressed almost exclusively in the adrenal cortex, and hence is the physiological ACTH receptor (3). After the MC2R gene was cloned (10), approximately 20 different MC2R mutations have been reported in patients with FGD (6, 7, 10). MC2R is a 297-amino acid protein, encoded by a gene on chromosome 18p11.2 "
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    ABSTRACT: Familial glucocorticoid deficiency (FGD) is a rare autosomal recessive disorder characterized by severe glucocorticoid deficiency associated with failure of adrenal responsiveness to ACTH but no mineralocorticoid deficiency. We report a 2 month-old boy of nonconsanguineous parents, presented with hyperpigmentation. Physical examination showed diffuse dark skin of body including, oral mucosa, gum, hands, nails and scrotum. Laboratory evaluation revealed low serum cortisol (0.3 microg/dL), with very high plasma ACTH level (18,000 pg/mL), and serum cortisol level did not increase after ACTH stimulation test. Serum sodium, potassium, plasma renin activity, aldosterone and 17-hydroxyprogesterone were normal. Sequence analysis of the ACTH receptor (MC2R) gene showed a homozygous mutation of D103N. Diagnosis of FGD was made and treatment started with oral hydrocortisone.
    Journal of Korean medical science 10/2009; 24(5):979-81. DOI:10.3346/jkms.2009.24.5.979 · 1.27 Impact Factor
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    • "As a consequence of the ACTH receptor-deficiency, these mutants failed to respond to ACTH with increased adenylyl cyclase activity (Schimmer, 1972; Schimmer and Tsao, 1984; Schimmer et al., 1987). These mutants have provided attractive models for studies of ACTH receptor structure–function relationships, due to the fact that they readily expressed functional receptors when transfected with genes encoding the ACTH receptor under the control of a strong promoter (Qiu et al., 1996; Elias et al., 1999; Fluck et al., 2002; Swords et al., 2002). "
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    ABSTRACT: The human adrenal cortex is a complex endocrine organ that secretes mineralocorticoids, glucocorticoids and adrenal androgens. These steroids arise from morphologically and biochemically distinct zones of the adrenal gland. Studying secretion of these distinct steroid hormones can make use of cells isolated from the adrenal gland but this requires animal sacrifice and the need for continued isolation for long-term studies. In addition primary cultures of adrenal cells have a limited life-span in culture and the cultured cells are often contaminated by the presence of non-steroidogenic cells. For that reason in vitro cell culture models have several benefits for research on adrenocortical function. Herein we discuss the available adrenocortical cell lines and their uses as model systems for adrenal studies. Focus is placed on the human NCI-H295 and mouse Y-1 adrenal cell lines, which have been used extensively as adrenocortical model systems. These cell lines have proven to be of considerable value in studying the molecular and biochemical mechanisms controlling adrenal steroidogenesis. The current review will discuss the attributes and limitations of the currently available adrenocortical cell lines as models for adrenal studies.
    Molecular and Cellular Endocrinology 01/2005; 228(1-2):23-38. DOI:10.1016/j.mce.2003.12.020 · 4.41 Impact Factor
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