The Natural Inverse Agonist Agouti-related Protein Induces Arrestin-mediated Endocytosis of Melanocortin-3 and -4 Receptors

Abstract

Agouti-related protein (Agrp), one of the two naturally occurring inverse agonists known to inhibit G protein-coupled receptor activity, regulates energy expenditure by decreasing basal and blocking agonist-promoted melanocortin receptor (MCR) signaling. Here we report that, in addition to its inverse agonistic activities, Agrp exhibits agonistic properties on the endocytosis pathway of melanocortin receptors. Sustained exposure of human embryonic kidney 293 cells to Agrp induced endocytosis of the MC3R or the MC4R. The extent and kinetics of Agrppromoted MCR endocytosis were similar to the endocytosis induced by melanocortins. Using the bioluminescence resonance energy transfer technique, we further showed that after binding of Agrp both MCRs interacted with ␤-arrestins. In line with this observation, in COS-7 cells co-expression of ␤-arrestins enhanced Agrp-induced MCR endocytosis, whereas in human embryonic kidney 293 cells co-transfection of ␤-arrestin-specific small interference RNAs diminished Agrp-promoted endocytosis. This new regulatory mechanism was likewise detectable in a cell line derived from murine hypothalamic neurons endogenously expressing MC4R, pointing to the physiological relevance of Agrp-promoted receptor endocytosis. In conclusion, we demonstrated that Agrp does not solely act by directly blocking MCR signaling but also by reducing the amount of MCR molecules accessible to melanocortins at the cell surface. This ␤-arrestin-dependent mechanism reveals a new aspect of MCR signaling in particular and refines the concept of G protein-coupled receptor antagonism in general.

Constitutive Cholesterol-dependent Endocytosis of Melanocortin-4 Receptor (MC4R) Is Essential to Maintain Receptor Responsiveness to α-Melanocyte-stimulating Hormone (α-MSH)

“…Together, the experiments of Fig. 4 indicate that, at least in unspecialized cells, ␤-arrestin and AP-2 function in MC4R internalization, consistent with previous reports (14,43). However, they do so at a reduced extent as compared with agonist-dependent internalization of ␤ 2 AR, suggesting differences in the mechanism by which two processes occur.…”

“…In this respect, overexpression of dominant-negative mutants of ␤-arrestin1-V53D in HEK 293 cells inhibited agonist-mediated disappearance of MC4R from the plasma membrane (14). In addition, silencing of ␤-arrestin1 and ␤-arrestin2 in the HEK 293 cells inhibited MC4R internalization in the presence of AgRP (43). Here, by using for the first time MEF with double knock-out of ␤-arrestin1 and ␤-arrestin2 to study MC4R traffic, we find that constitutive internalization of receptor is ␤-arrestin-dependent but to a lower extent (3-fold) than that of ␤ 2 AR.…”

“…Others have found that internalization of MC4R in the presence of agonist and antagonist is dependent on ␤-arrestin (14,43). In this respect, overexpression of dominant-negative mutants of ␤-arrestin1-V53D in HEK 293 cells inhibited agonist-mediated disappearance of MC4R from the plasma membrane (14).…”

“…The ␤-arrestin bound to the GPCR interacts with both AP-2 and clathrin to internalize ␤ 2 AR (39, 41, 42). Similarly, it has been found that MC4R internalization in the presence of ␣-MSH and AgRP is also dependent on ␤-arrestin in HEK 293 cells and COS-7 cells (14,43). Here, we tested whether MC4R internalization, in the absence of agonist, is also dependent on ␤-arrestin.…”

Melanocortin receptors and their accessory proteins

“…The release of proopiomelanocortin (POMC)-derived peptides from neurons in the arcuate nucleus, which are stimulated by leptin results in the activation of MC4R signaling in the PVN (Cheung et al, 1997;Schwartz et al, 1997). Uniquely for a biological system, a physiologically relevant endogenous antagonist and inverse agonist of MC4R (as well as MC3R) signaling is also produced in the hypothalamus, Frontiers in Endocrinology | Neuroendocrine Science agouti-related peptide (AGRP; Nijenhuis et al, 2001;Breit et al, 2006). ARGP is produced by leptin responsive neurons in the arcuate nucleus that are inhibited by leptin.…”