Arginine 313 of the putative 8th helix mediates Gα_q/14 coupling of human CC chemokine receptors CCR2a and CCR2b.

In man, two CC chemokine receptor isoforms, CCR2a and CCR2b, are present that belong to the rhodopsin-like G protein-coupled receptor family, and couple to G_i and G_q family members. The CCR2 receptors are known to regulate canonical functions of chemokines such as directed migration of leukocytes, and to potentially control non-canonical functions such as differentiation, proliferation, and gene transcription of immune and non-immune cells. We recently reported on the activation of phospholipase C isoenzymes and RhoA GTPases by coupling of the two CCR2 receptors to members of the G_q family, in particular Gα_q and Gα₁₄. So far little is known about the structural requirements for the CCR2/G_q/14 interaction. Interestingly, the CCR2 receptor isoforms are identical up to arginine 313 (R³¹³) that is part of the putative 8th helix in CCR2 receptors, and the 8th helix has been implicated in the interaction of rhodopsin-like G protein-coupled receptors with Gα_q. In the present work we describe that the 8th helix of both CCR2a and CCR2b is critically involved in selectively activating Gα_q/14-regulated signaling. Refined analysis using various CCR2a and CCR2b mutants and analyzing their cellular signaling, e.g. ligand-dependent (i) activation of phospholipase C isoenzymes, (ii) stimulation of serum response factor-mediated gene transcription, (iii) activation of mitogen-activated protein kinases, (iv) internalization, and (v) changes in intracellular calcium concentrations, identified arginine 313 within the amino terminal portion of helix 8 to play a role for the agonist-mediated conformational changes and the formation of a Gα_q/14 binding surface. We show that R³¹³ determines Gα_q/14 protein-dependent but not G_i protein-dependent cellular signaling, and plays no role in G_q/G_i-independent receptor internalization, indicating a role of R³¹³ in biased signaling of CCR2 receptors.

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