Characterization of corticotropin receptors on adrenocortical cells.

Abstract

The binding ofcorticotropin (ACTH) to receptors on isolated rat adrenocortical cells was investigated with the aid of [['25I]ITyro, Phe2, Nle4]ACTH-(1-38) (1'I-ACTH analog) which retained full biological potency and had a specific radioactivity of 1800 ± 75 Ci/mmol. Binding was highly specific to adrenocortical cells, and the radioactive peptide was displaced by low concentrations ofACTH but not by other basic peptides. Bind Despite numerous attempts to investigate corticotropin (ACTH) receptors by direct binding methods, detection and characterization ofthe physiologically relevant receptors for the hormone has proved to be a formidable task. The two major difficulties encountered in binding studies with ACTH are the extraordinary propensity of the hormone to bind to inert materials and nonreceptor components of the target tissue, and the low biological potencies of the radiolabeled ACTH preparations generally used in binding studies (1-4).The "2I-labeled ACTH (125I-ACTH) preparations used by Lefkowitz et aL (1) and Ontjes et aL (3) had biological activities of32 and 15 units/mg, respectively, compared to a value of200 units/mg for unmodified ACTH reported by Rae and Schimmer (5), as measured by the stimulation of adenylate cyclase in adrenocortical cell membrane fractions. McIlhinney and Schulster (6, 7) prepared '"I-ACTH having 50% of the steroidogenic activity of the native hormone by the lactoperoxidase method.Structure-function studies (5,8,9) have shown that the loss of biological potency of ACTH results from the introduction of a bulky iodine atom into the tyrosine residue in position 2 and the oxidation of the methionine residue in position 4. We recently circumvented these difficulties by synthesizing an analog of the hormone in which the tyrosine in position 2 is replaced by phenylalanine and the methionine in position 4 is replaced by norleucine (10). This peptide, referred to hereafter as ACTH analog, was shown to be equipotent with ACTH in stimulating steroidogenesis in adrenocortical cells. We have also shown that iodination of the ACTH analog and purification by reversephase high-pressure liquid chromatography (HPLC) yields 125I-ACTH analog with the theoretically attainable specific radioactivity and full biological potency (11). By the use of this radioligand we have succeeded in identifying the specific receptors for the hormone on rat adrenocortical cells. MATERIALS AND METHODS125I-ACTH Analog. [Phe2, Nle4]ACTH-(1-38) was synthesized by the solid-phase method and purified as described (10).[[251I]ITyr23, Phe2, Nle4]ACTH-(1-38), the 125I-ACTH analog, was prepared by the chloramine-T method and separated from free iodide by gel filtration on Sephadex LH-20 as described (11). It was stored at 4°C and freshly purified by reverse-phase HPLC for each experiment (11). The HPLC solvents were removed by lyophilization to 1/10th the original volume and the labeled peptide was diluted with medium 199 (GIBCO) containing 0.5% bovine serum albumin purified as described (12), and 0.01% bacitracin (A...

Differential expression of VEGF receptors in adrenal atrophy induced by dexamethasone: a protective role of ACTH

“…Our results suggest that ACTH is the major regulator of these effects, since ACTH injections within 7 days restored plasma corticosterone level to control values. These results are in agreement with studies in the hypophysectomized fetal sheep, which showed that adrenal growth could be restored by in utero ACTH replacement therapy (6,8,24).…”

“…These fenestrations are the pores through which releasing factors within the hemal milieu can access the cells. In adult mammals, adrenal growth and steroidogenesis are regulated by adrenocorticotropin (ACTH) (27), which acts through interaction with a specific melanocortin receptor (MC2R) specifically expressed in the adrenal cortex (6,32). This guanine nucleotide-binding protein-coupled receptor elicits the activation of adenylate cyclase and the production of cAMP, which activate the protein kinase A-signaling pathway (39,53) and the normal functioning of adrenocortical cells.…”

60 YEARS OF POMC: Melanocortin receptors: evolution of ligand selectivity for melanocortin peptides

“…Before the characterization of mouse MC2R by Mountjoy et al (1992), studies in the 1970s and 1980s on the activation of the 'ACTH' receptor on mammalian adrenal cortex cells found that these cells could be stimulated by ACTH, but not by α-MSH (Schwyzer 1977). In fact, α-MSH could not competitively inhibit the binding of ACTH to the 'ACTH' receptor (Buckley & Ramachandran 1981). The critical difference between ACTH and α-MSH is the presence of the tetrabasic amino acid motif (KKRRP; Fig.…”

MOLECULAR EVOLUTION OF GPCRS: Melanocortin/melanocortin receptors

“…inability of a-MSH to even bind to mammalian MC2R (Buckley & Ramachandran 1981, Mountjoy et al 1992 are direct outcomes of this interaction. Studies on mammalian MRAP1 and MC2R orthologs have revealed a number of features of the MC2R/MRAP1 interaction and have raised several new questions.…”