Chemokine receptor (CKR) signaling forms the basis of essential immune cellular functions, and dysregulated CKR signaling underpins numerous disease processes of the immune system and beyond. CKRs, which belong to the seven transmembrane domain receptor (7TMR) superfamily, initiate signaling upon binding of endogenous, secreted chemokine ligands. Chemokine-CKR interactions are traditionally described by a two-step/two-site mechanism, in which the CKR N-terminus recognizes the chemokine globular core (i.e. site 1 interaction), followed by activation when the unstructured chemokine N-terminus is inserted into the receptor TM bundle (i.e. site 2 interaction). Several recent studies challenge the structural independence of sites 1 and 2 by demonstrating physical and allosteric links between these supposedly separate sites. Others contest the functional independence of these sites, identifying nuanced roles for site 1 and other interactions in CKR activation. These developments emerge within a rapidly changing landscape in which CKR signaling is influenced by receptor PTMs, chemokine and CKR dimerization, and endogenous non-chemokine ligands. Simultaneous advances in the structural and functional characterization of 7TMR biased signaling have altered how we understand promiscuous chemokine-CKR interactions. In this review, we explore new paradigms in CKR signal transduction by considering studies that depict a more intricate architecture governing the consequences of chemokine-CKR interactions.

These results suggest a different interaction mechanism between this atypical receptor and its ligands and illustrate its strong propensity to activation.

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Recent observations suggest that atypical chemokine receptor (ACKR)3 and chemokine (C-X-C motif) receptor (CXCR)4 regulate human vascular smooth muscle function through hetero-oligomerization with α1-adrenoceptors. Here, we show that ACKR3 also regulates arginine vasopressin receptor (AVPR)1A. We observed that ACKR3 agonists inhibit arginine vasopressin (aVP)-induced inositol trisphosphate (IP3) production in human vascular smooth muscle cells (hVSMCs) and antagonize aVP-mediated constriction of isolated arteries. Proximity ligation assays, co-immunoprecipitation and bioluminescence resonance energy transfer experiments suggested that recombinant and endogenous ACKR3 and AVPR1A interact on the cell surface. Interference with ACKR3 : AVPR1A heteromerization using siRNA and peptide analogues of transmembrane domains of ACKR3 abolished aVP-induced IP3 production. aVP stimulation resulted in β-arrestin 2 recruitment to AVPR1A and ACKR3. While ACKR3 activation failed to cross-recruit β-arrestin 2 to AVPR1A, the presence of ACKR3 reduced the efficacy of aVP-induced β-arrestin 2 recruitment to AVPR1A. AVPR1A and ACKR3 co-internalized upon agonist stimulation in hVSMC. These data suggest that AVPR1A : ACKR3 heteromers are constitutively expressed in hVSMC, provide insights into molecular events at the heteromeric receptor complex, and offer a mechanistic basis for interactions between the innate immune and vasoactive neurohormonal systems. Our findings suggest that ACKR3 is a regulator of vascular smooth muscle function and a possible drug target in diseases associated with impaired vascular reactivity.

CXCL12 is a human chemokine that recognizes the CXCR4 receptor and is involved in immune responses and metastatic cancer. Interactions between CXCL12 and CXCR4 are an important drug target but, like other elongated protein-protein interfaces, present challenges for small molecule ligand discovery due to the relatively shallow and featureless binding surfaces. Calculations using an NMR complex structure revealed a binding hot spot on CXCL12 that normally interacts with the I4/I6 residues from CXCR4. Virtual screening was performed against the NMR model, and subsequent testing has verified the specific binding of multiple docking hits to this site. Together with our previous results targeting two other binding pockets that recognize sulfotyrosine residues (sY12 and sY21) of CXCR4, including a new analog against the sY12 binding site reported herein, we demonstrate that protein-protein interfaces can often possess multiple sites for engineering specific small molecule ligands that provide lead compounds for subsequent optimization by fragment based approaches.