Alanine Scanning Mutagenesis of the DRYxxI Motif and Intracellular Loop 2 of Human Melanocortin-4 Receptor

Abstract

The melanocortin-4 receptor (MC4R) is a member of the G-protein-coupled receptor (GPCR) superfamily, which has been extensively studied in obesity pathogenesis due to its critical role in regulating energy homeostasis. Both the Gs-cAMP and ERK1/2 cascades are known as important intracellular signaling pathways initiated by the MC4R. The DRYxxI motif at the end of transmembrane domain 3 and the intracellular loop 2 (ICL2) are thought to be crucial for receptor function in several GPCRs. To study the functions of this domain in MC4R, we performed alanine-scanning mutagenesis on seventeen residues. We showed that one residue was critical for receptor cell surface expression. Eight residues were important for ligand binding. Mutations of three residues impaired Gs-cAMP signaling without changing the binding properties. Investigation on constitutive activities of all the mutants in the cAMP pathway revealed that six residues were involved in constraining the receptor in inactive states and five residues were important for receptor activation in the absence of an agonist. In addition, mutations of four residues impaired the ligand-stimulated ERK1/2 signaling pathway without affecting the binding properties. We also showed that some mutants were biased to the Gs-cAMP or ERK1/2 signaling pathway. In summary, we demonstrated that the DRYxxI motif and ICL2 were important for MC4R function.

Structures of active melanocortin-4 receptor–Gs-protein complexes with NDP-α-MSH and setmelanotide

“…S18 ). In agreement with the essential role of IL2 and position 3.58 in Gs-coupling, previously investigated mutants L155A, R147A, T150A, and Y157A have been shown to eliminate any basal signaling activity of MC4R, 45 presumably by disturbing the interface contacts between the receptor and its effector protein.…”

Structures of active melanocortin-4 receptor−Gs-protein complexes with NDP-α-MSH and setmelanotide

“…Yet, there is an additional water-mediated stabilization to A154 IL2-3.57 in IL2 (Figure 6E). Interestingly, the H158A mutation leads to constitutive receptor activation and causes a slight increase in agonist-induced Gs-signaling (Figure 4D-4E, Tables S7 and S8 and (Yang and Tao, 2020)) in agreement with the pathogenic gain-of-function mutant H158R (Hinney et al, 2006). Compared to the antagonized MC4R structure without structurally visible interaction of H158, it is not yet obvious why mutants at H158 lead to constitutive receptor activation.…”

“…Of note, a large number of class A GPCRs display a leucine or isoleucine at the IL2-3.58 position that often exists in combination with a tyrosine in IL2 (IL2-3.60) (Figure S20). In agreement with the essential role of IL2 and position 3.58 for Gs-coupling, previously investigated mutants L155A, R147A, T150A, and Y157A eliminate any basal signaling activity of MC4R (Yang and Tao, 2020), presumably by disturbing the interface contacts between the receptor and its effector protein.…”

“…Compared to the antagonized MC4R structure without structurally visible interaction of H158, it is not yet obvious why mutants at H158 lead to constitutive receptor activation. However, in the context of the known constitutively activating mutants D146A, F149A, and F152A (Yang and Tao, 2020) the importance of the entire IL2− TM3 transition as a key-switch for regulation of MC4R-signaling and G-protein engagement is highlighted.…”

Hydrophilic molecularly imprinted polymers functionalized magnetic carbon nanotubes for selective extraction of cyclic adenosine monophosphate from winter jujube

“…Cyclic adenosine monophosphate (cAMP), is an important substance involved in the regulation of cellular biological function and material metabolism. As the second messenger in the life information transmission [1], cAMP also plays a crucial role in many biological processes such as the regulation of enzyme activity [2], gene expression [3,4], and cell proliferation and differentiation [5]. As a bioactive substance, cAMP has numerous health-promoting functions including anticancer [6], antiallergic [7], anti-inflammatory [8], anti-insomnia [9], improving human immunity [10], etc.…”