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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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The melanocortin 2 receptor (MC2R) accessory protein (MRAP) is a small single-transmembrane domain protein that plays a pivotal role in the function of the MC2R. The pituitary hormone, ACTH, acts via this receptor complex to stimulate adrenal steroidogenesis. Using both coimmunoprecipitation and bioluminescence resonance energy transfer (BRET), we show that the MC2R is constitutively homodimerized in cells. Furthermore, consistent with previous data, we also show that MRAP exists as an antiparallel homodimer. ACTH enhanced the BRET signal between MC2R homodimers as well as MC2R-MRAP heterodimers. However, ACTH did not enhance the physical interaction between these dimers as determined by coimmunoprecipitation. Real-time BRET analysis of the MRAP-MC2R interaction revealed two distinct phases of the ACTH-dependent BRET increase, an initial complex series of changes occurring over the first 2 min and a later persistent increase in BRET signal. The slower ACTH-dependent phase was inhibited by the protein kinase A inhibitor KT5720, suggesting that signal transduction was a prerequisite for this later conformational change. The MRAP-MC2R BRET approach provides a unique tool with which to analyze the activation of this receptor.

Receptor desensitization provides a potential mechanism for the regulation of adrenocortical adrenocorticotropin (ACTH) responsiveness. Using the mouse adrenocortical Y1 cell line we demonstrate that ACTH effectively desensitizes the cAMP response of its own receptor, the melanocortin 2 receptor (MC2R), in these cells with a maximal effect between 30 and 60 min. Neither forskolin nor isoproterenol (in Y1 cells stably transfected with the beta(2)-adrenergic receptor) desensitize this ACTH response. ACTH desensitizes its receptor at concentrations at which only a fraction of receptors are occupied, implying that this mechanism acts on agonist-unoccupied receptors. Y1 cells express G protein-coupled receptor kinase (GRK) 2 and 5, but stable expression of a dominant negative GRK2 (K220W) only marginally reduces the desensitization by ACTH. The protein kinase A (PKA) inhibitor, H89, extinguishes almost the entire desensitization response over the initial 30-min period at all concentrations of ACTH. A mutant MC2R in which the single consensus PKA phosphorylation site has been mutated (S208A) when expressed in MC2R-negative Y6 cells is also unable to desensitize. These data imply a heterologous, PKA-dependent, mode of desensitization, which is restricted to agonist-occupied and -unoccupied MC2R, possibly as a consequence of receptor/effector complexes that functionally compartmentalize this receptor.

Melanocortins are known to be involved in the regulation of feeding behavior. These hormones mediate their effects through G-protein-coupled receptors by stimulating adenylate cyclase. In this study we describe the functional response of melanocortin 4 receptor (MC4R) and melanocortin 3 receptor (MC3R) in HEK 293T cells, by using a luciferase reporter gene under the transcriptional control of a cAMP-responsive element (CRE) as a monitor of intracellular cAMP levels and cAMP-regulated gene expression. We were able to show that MC4R and MC3R expressed in the human cell line HEK 293T stimulate transcription induced by stimulation with different analogs of alpha-melanocyte-stimulating hormone (alpha-MSH) at different levels. In our assay of CRE-mediated gene transcription activity, alpha-MSH-ND was the most efficient alpha-MSH analog for MC4R whereas NDP-MSH was the most efficient for MC3R. Changing the His6 residue of alpha-MSH-ND to Gln or Lys markedly decreased CRE-mediated luciferase activity for MC3R compared with MC4R. On analysis by modeling the receptor-ligand complex by NMR, [Gln6]alpha-MSH-ND and [Lys6]alpha-MSH-ND showed different conformational interactions between MC3R and MC4R. Furthermore, the maximum coupling efficiency of MC4R and MC3R to G proteins was different; MC4R showed only 30-50% of the maximum activity induced by MC3R. In total, our results suggest that a differential receptor-ligand interaction is involved and that the relative interactions of MC3R and MC4R with G protein are possibly quantitatively and qualitatively different.