The E92K Melanocortin 1 Receptor Mutant Induces cAMP Production and Arrestin Recruitment but Not ERK Activity Indicating Biased Constitutive Signaling

BACKGROUND: The melanocortin 1 receptor (MC1R) constitutes a key regulator of melanism. Consequently, many naturally-occurring MC1R mutations are associated with a change in color. An example is the Glu-to-Lys substitution found at position II:20/2.60 in the top of transmembrane helix II which has been identified in melanic mice and several other species. This mutation induces a pronounced increase in MC1R constitutive activity suggesting a link between constitutive activity and melanism which is corroborated by the attenuation of α-melanocyte stimulating hormone (αMSH) induced activation. However, the mechanism by which the mutation induces constitutive activity is currently not known. METHODOLOGY/PRINCIPAL FINDINGS: Here we characterize the constitutive activity, cell surface expression and internalization of the mouse mutant, Mc1r E92K. As previously reported, only positively charged residues at position II:20/2.60 induced an increase in constitutive activity as measured by cAMP accumulation and CREB activation. Furthermore, the mutation induced a constitutive recruitment of β-arrestin. This phenomenon is only observed in MC1R, however, as the equivalent mutations in MC2-5R had no effect on receptor signaling. Interestingly, the mutation did not induce constitutive ERK1/2 phosphorylation or increase the internalization rate indicating the constitutive activity to be biased. Finally, to identify regions of importance for the increased constitutive activity of Mc1r E92K, we employed a chimeric approach and identified G102 and L110 in the extracellular loop 1 to be selectively important for the constitutive activity as this, but not αMSH-mediated activation, was abolished upon Ala substitution. CONCLUSIONS/SIGNIFICANCE: It is concluded that the E92K mutation induces an active conformation distinct from that induced by αMSH and that the extracellular loop 1 is involved in maintaining this conformational state. In turn, the results suggest that in MC1R, which lacks an extracellular loop 2, the first extracellular loop may play a more prominent role during receptor activation than in general

Oxysterol-EBI2 signaling in immune regulation and viral infection

The 7TM G protein-coupled receptor EBI2 (GPR183) was identified in 1993 by its substantial upregulation in Epstein-Barr Virus (EBV) infected cells. It is primarily expressed in lymphoid cells; most abundantly in B lymphocytes. EBI2 is central for the positioning of B cells within the lymphoid organs, a process that is regulated in part by a chemotactic gradient formed by the endogenous lipid agonists, and in part by a fine-tuned regulation of EBI2 cell surface expression. The most potent endogenous agonist is 7a, 25-dihydroxyxcholesterol (7a,25-OHC), yet EBI2 binds many structurally related oxysterols in a pocket defined by the upper parts of the transmembrane helices and extracellular receptor regions. It signals via Gi and also G protein-independent pathways like b-arrestin recruitment. The concerted action of these pathways leads to cell migration. By using genetically engineered up- and down regulation, EBI2 was recently shown also to induce cell proliferation; an action that could be inhibited by small molecule antagonists. Here we focus on the EBI2/oxysterol axis in immune control, hereunder the role in the EBV lifecycle. We also summarize the structural and functional properties of EBI2 interaction with oxysterol agonists and small molecule antagonist and ultimately discuss the drugability of EBI2 for diseases within the immune system

Oxysterol‐EBI2 signaling in immune regulation and viral infection

The seven transmembrane G protein-coupled receptor Epstein-Barr virus (EBV) induced gene 2 (EBI2; also known as GPR183) was identified in 1993 on the basis of its substantial upregulation in EBV-infected cells. It is primarily expressed in lymphoid cells; most abundantly in B cells. EBI2 is central for the positioning of B cells within the lymphoid organs, a process that is regulated in part by a chemotactic gradient formed by the endogenous lipid agonists, and in part by a fine-tuned regulation of EBI2 cell surface expression. The most potent endogenous EBI2 agonist is 7α, 25-dihydroxyxcholesterol (7α,25-OHC), yet many structurally related oxysterols can bind to an EBI2 pocket that is defined by the upper parts of the transmembrane helices and extracellular receptor regions. EBI2 signals via Gαi, as well as via G protein-independent pathways like β-arrestin recruitment. The concerted action of these pathways leads to cell migration. By genetically interfering with its up- and downregulation, EBI2 was also recently shown to induce cell proliferation, an action that could be inhibited by small molecule antagonists. Here, we focus on the oxysterol–EBI2 axis in immune control, including its role in the EBV life cycle. We also summarize the structural and functional properties of EBI2 interaction with oxysterol agonists and small molecule antagonists and discuss EBI2 as therapeutic target for diseases of the immune system

Oxysterol-EBI2 signaling in immune regulation and viral infection

The 7TM G protein-coupled receptor EBI2 (GPR183) was identified in 1993 by its substantial upregulation in Epstein-Barr Virus (EBV) infected cells. It is primarily expressed in lymphoid cells; most abundantly in B lymphocytes. EBI2 is central for the positioning of B cells within the lymphoid organs, a process that is regulated in part by a chemotactic gradient formed by the endogenous lipid agonists, and in part by a fine-tuned regulation of EBI2 cell surface expression. The most potent endogenous agonist is 7a, 25-dihydroxyxcholesterol (7a,25-OHC), yet EBI2 binds many structurally related oxysterols in a pocket defined by the upper parts of the transmembrane helices and extracellular receptor regions. It signals via Gi and also G protein-independent pathways like b-arrestin recruitment. The concerted action of these pathways leads to cell migration. By using genetically engineered up- and down regulation, EBI2 was recently shown also to induce cell proliferation; an action that could be inhibited by small molecule antagonists. Here we focus on the EBI2/oxysterol axis in immune control, hereunder the role in the EBV lifecycle. We also summarize the structural and functional properties of EBI2 interaction with oxysterol agonists and small molecule antagonist and ultimately discuss the drugability of EBI2 for diseases within the immune system