Functional Selectivity of Melanocortin 4 Receptor Peptide and Nonpeptide Agonists: Evidence for Ligand-Specific Conformational States

Abstract

Agonists of the melanocortin 4 (MC4) receptor have potential pharmaceutical benefit in the treatment of obesity and sexual dysfunction. In this study, we have compared the ability of a number of peptide and nonpeptide agonists to activate a FLAG-tagged human MC4 (FMC4) receptor, as measured by both cAMP accumulation and calcium mobilization using a fluorometric imaging plate reader (FLIPR). In addition, we have analyzed the ability of these agonists to cause receptor internalization, as measured by fluorescence-activated cell sorting analysis. The endogenous agonist ␣-melanocortin-stimulating hormone (␣-MSH) increased cAMP accumulation, calcium mobilization, and receptor internalization in a dose-dependent manner in human embryonic kidney 293 cells expressing the FMC4 receptor. The activity of the other agonists varied considerably in these assays, and overall, the potency and intrinsic activity of the agonists in the cAMP accumulation assays did not correlate with their potency or intrinsic activity in either the FLIPR or receptor internalization assays. Agonists could be clearly separated into two functional classes based on their structure. Peptide agonists ␤-MSH, des-acetyl-␣-MSH, and [Nle 4 , D-Phe 7 ]-␣-melanocortin-stimulating hormone exhibited 80 to 112% of the maximal ␣-MSH response in cAMP accumulation and 62 to 96% in FLIPR assays and were able to cause 75 to 118% of receptor internalization induced by ␣-MSH. Conversely, although the nonpeptide agonists exhibited 73 to 149% of the ␣-MSH response in the cAMP accumulation assays, they were significantly impaired in the FLIPR (7-40%) and receptor internalization (Ϫ5-38%) assays. These findings demonstrate an important difference in activation and internalization of the MC4 receptor by nonpeptide versus peptide agonists and provides evidence of agonist-specific conformational states.The melanocortin 4 receptor (MC4R) is a member of the seven transmembrane receptor family that initiate signal transduction through activation of heterotrimeric G proteins. There are four additional members of the melanocortin receptor subfamily, distinguishable by their distribution and physiology. Melanocortin receptors are all activated by one or more pro-opiomelanocortin-derived peptides, which include ␣-, ␤-, and ␥-melanocortin-stimulating hormone (MSH). In addition, the melanocortin receptors are regulated by the antagonists/inverse agonists, AGRP and agouti (Lu et al., 1994;Ollmann et al., 1997).The melanocortin receptors all signal through G␣ s , which stimulates the production of cAMP by adenylate cyclase. The MC3 receptor also functions through phospholipase C to increase intracellular calcium (Konda et al., 1994). Likewise, the MC4 receptor mediates an increase in intracellular calcium, but reportedly through a cholera toxin-sensitive pathway rather than phospholipase C (Mountjoy et al., 2001).

Biased signaling at neural melanocortin receptors in regulation of energy homeostasis

“…Biased signaling, as an important concept in GPCR intracellular signaling, has been identified in many members of GPCR family [97, 98]. Data from us and others [45, 47, 51, 52, 56] suggested that neural MCRs are able to selectively stabilize particular receptor active conformations and preferentially trigger distinct signaling pathways, consistent with the multi-state model of receptor activation and biased signaling in other GPCRs.…”

“…However, increasing number of recent studies have demonstrated that similar to other GPCRs, neural MCRs can couple to other G proteins or signaling mediators to trigger the downstream signaling pathways, in addition to the conventional Gαs-cAMP signaling pathway. Biased signaling was also proposed for neural MCRs since we and others identified some biased mutant neural MCRs which selectively trigger signaling pathways upon ligand stimulation, and some biased agonists which can selectively induce signaling pathways through neural MCRs [45-56]. …”

Functional Selectivity of NK1Receptor Signaling: Peptide Agonists Can Preferentially Produce Receptor Activation or Desensitization

“…Functional selectivity has been well described among amine receptors including serotonergic (Roth and Chuang, 1987;Berg et al, 1998), dopaminergic (Lewis et al, 1998;Gay et al, 2004), and adrenergic (Jim et al, 1985) receptors. Ligand functional selectivity has also been demonstrated for several other peptide receptors (Hunyady et al, 1994;Whistler et al, 1999;Zhang et al, 1999;Kapusta et al, 2005;McLaughlin et al, 2005;Nickolls et al, 2005).…”

Functional selectivity – chance for better and safer drugs?

“…functionally selective agonists. Biased agonism, has been identified for many therapeutically important GPCRs such as α 1 -and β-adrenergic receptors [16,[23][24][25], μ-opioid receptors (MOR) [26,27], cannabinoid receptors [28,29], D 2L and D 1 dopamine receptors [30][31][32][33], serotonin receptors [17,34,35], chemokine receptor CCR7 [36,37], gonadotropin-releasing hormone receptors [38,39], parathyroid hormone 1 receptors (PTH1R) [40,41], angiotensin 1A (AT 1A ) receptors [42] or MC4 melanocortin receptors [43].…”