Engineering the Melanocortin-4 Receptor to Control Constitutive and Ligand-Mediated Gs Signaling In Vivo

Abstract

The molecular and functional diversity of G protein–coupled receptors is essential to many physiological processes. However, this diversity presents a significant challenge to understanding the G protein–mediated signaling events that underlie a specific physiological response. To increase our understanding of these processes, we sought to gain control of the timing and specificity of Gs signaling in vivo. We used naturally occurring human mutations to develop two Gs-coupled engineered receptors that respond solely to a synthetic ligand (RASSLs). Our Gs-coupled RASSLs are based on the melanocortin-4 receptor, a centrally expressed receptor that plays an important role in the regulation of body weight. These RASSLs are not activated by the endogenous hormone α-melanocyte-stimulating hormone but respond potently to a selective synthetic ligand, tetrahydroisoquinoline. The RASSL variants reported here differ in their intrinsic basal activities, allowing the separation of the effects of basal signaling from ligand-mediated activation of the Gs pathway in vivo. These RASSLs can be used to activate Gs signaling in any tissue, but would be particularly useful for analyzing downstream events that mediate body weight regulation in mice. Our study also demonstrates the use of human genetic variation for protein engineering.

In Vivo Evidence for Inverse Agonism of Agouti-Related Peptide in the Central Nervous System of Proopiomelanocortin-Deficient Mice

“…Supporting this possibility are recent reports that the human MC4-R undergoes ligand-independent cycles of endo-and exocytosis in transfected neuroblastoma cells and immortalized hypothalamic neurons (48) and that antibodies directed against an epitope in the NH 2 -terminal domain of the MC4-R act as inverse agonists in cell lines and intact animal models (49). Assessment of the effects of AgRP in knock-in mouse models containing humanized MC4-R with the inactivating NH 2 -terminal mutations or other characterized, constitutively active receptors that respond solely to a synthetic ligand (50) would be useful to confirm that the mechanism of action of AgRP is partly mediated through modulation of MC4-R constitutive activity. …”

Structural Motifs of Importance for the Constitutive Activity of the Orphan 7TM Receptor EBI2: Analysis of Receptor Activation in the Absence of an Agonist

“…In the absence of agonists, the receptor activity was correlated to cell surface expression determined by ELISA using antibodies against the M1-FLAG-tag (Figs. 3 and 5-7)-a method also used in other studies (Govaerts et al, 2001b;Srinivasan et al, 2007). Among the multiple receptor DNA concentrations analyzed (Fig.…”

Remote Control of Neuronal Signaling

“…An F435A mutation reduced the affinity and potency (25-and 200-fold, respectively) of histamine yet increased the affinity and potency of the synthetic phenylhistamines (54-and 2600-fold, respectively). Srinivasan et al (2003Srinivasan et al ( , 2007 developed the first RASSLs to signal through G s . They introduced into the melanocortin-4 (MC4) receptor naturally occurring mu-CELL-TYPE CONTROL OF SIGNALING tations that abolish activity of the cognate ligand ␣-melanocyte-stimulating hormone.…”

“…First, although the receptors are activated solely by synthetic ligands, the synthetic ligands do not solely activate the designer receptors. For example, spiradoline has only 200-fold higher potency at Ro1 than at hKOR (Coward et al, 1998), and THIQ is more potent at MC4 than at Rm1 or Rm2 (Srinivasan et al, 2007). Therefore, to selectively activate only the RASSL and not the native receptor in vivo, one must either use a knockout background, which introduces a variety of confounds into the experimental design, or one must locally infuse the synthetic ligand to a tissue or region that expresses only the RASSL and not the wildtype receptor.…”