Abstract
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.
Highlights
Receptor desensitization provides a potential mechanism for the regulation of adrenocortical adrenocorticotropin (ACTH) responsiveness
The existence of other melanocortin receptors in these cells was excluded by demonstrating that norleucine4 D-phenylalanine7-melanocyte-stimulating hormone (NDP-MSH), a superagonist peptide that stimulates all of the melanocortin receptors except the melanocortin 2 receptor (MC2R), failed to generate a cAMP response
Earlier studies have shown that the desensitization of ACTHstimulated adenylate cyclase activity is readily detectable in Y1 cells and tends to be a more prominent phenomenon than the loss of cell surface binding observed after prolonged ACTH stimulation [14, 15]
Summary
Materials—Cell culture reagents were purchased from SigmaAldrich (Dorset, UK) and Life Technologies, Inc. (Paisley, Scotland, UK). CAMP Measurement—Cells were seeded in 6-well plates 24 – 48 h prior to stimulation with ACTH-(1–24) and incubated in serum-free medium for at least 1 h prior to stimulation. Cells were stimulated in the presence of 1 mM 3-isobutyl-1-methylxanthine (IBMX) for 30 min at 37 °C. Cells were heated at 100 °C for 5 min, centrifuged to precipitate cell debris, and the supernatant was stored for assay. The cells were washed with serum-free medium and restimulated with ACTH (10Ϫ8 M) in the presence of IBMX (1 mM) for 30 min in this collection phase. Prior to use the total cell lysate was heated to 85 °C for 10 min and reconstituted using a fine needle attached to a syringe. Statistical significance was determined by Student’s t test or by two-way analysis of variance using MS Excel
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