<p>Poster presented at the 13th annual meeting of the Great Lakes GPCR Retreat. This international meeting was held on October 17th, 2012 in London, ON.</p>
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<p><strong>ABSTRACT</strong></p>
<p>Constitutively active GPCR mutants (CAMs) spontaneously activate G-protein signaling and are models of agonist-induced GPCR activation. Constitutively inactive mutants (CIMs) that spontaneously recruit beta-arrestin are devoid of G-protein signaling and are models of agonist-induced GPCR desensitization. Inverse agonists have negative efficacy on CAM activity. Under the perspective of biased agonism, it is conceivable that CAM inverse agonists (of G-protein signaling) do or do not act as inverse agonists (of arrestin recruitment) on CIMs. CXCR4 a G-protein coupled receptor (GPCR) that binds the chemokine ligand CXCL12 (SDF-1α). CXCR4 is involved in different pathologies including tumor metastasis, inflammatory diseases, and HIV infection. It is the most widely expressed chemokine receptor on cancer cells. After activation, CXCR4 signals through two major subsequent pathways: Gα/i and β-arrestin. β-arrestins terminate G-protein signaling and target the desensitized receptor to endocytosis. TC14012 is known as an inverse agonist of CXCR4 on the canonical G-protein pathways, while AMD3100 is a neutral antagonist. Though the role of β-Arrestins in the CXCR4 signaling is well established, the effect of TC14012 or AMD3100 was not investigated on this pathway so far. This is mainly due to the lack of a suitable model (CXCR4 CIM). We tested a set of CXCR4 mutants on their spontaneous signaling capacity of Gα/i and β-Arrestin2 pathways, using BRET2 as a cAMP reporter (G-protein pathway) and to detect the recruitment of β-Arrestin2 to the receptor. Besides the already reported N119S CAM, we found that R134A was a CIM that was inactive on the Gα/i signaling, but constitutively recruited arrestin. TC14012, but not AMD3100, was found to be an inverse agonist on the β-Arrestin2 pathway. Their effects on CIM R134A surface expression and erk kinase activation remain to be determined.</p
