The chemokine X-factor: Structure-function analysis of the CXC motif at CXCR4 and ACKR3.

The human chemokine family consists of 46 protein ligands that induce chemotactic cell migration by activating a family of 23 G protein-coupled receptors. The two major chemokine subfamilies, CC and CXC, bind distinct receptor subsets. A sequence motif defining these families, the X position in the CXC motif, is not predicted to make significant contacts with the receptor, but instead links structural elements associated with binding and activation. Here, we use comparative analysis of chemokine NMR structures, structural modeling, and molecular dynamic simulations that suggested the X position reorients the chemokine N terminus. Using CXCL12 as a model CXC chemokine, deletion of the X residue (Pro-10) had little to no impact on the folded chemokine structure but diminished CXCR4 agonist activity as measured by ERK phosphorylation, chemotaxis, and Gi/o-mediated cAMP inhibition. Functional impairment was attributed to over 100-fold loss of CXCR4 binding affinity. Binding to the other CXCL12 receptor, ACKR3, was diminished nearly 500-fold. Deletion of Pro-10 had little effect on CXCL12 binding to the CXCR4 N terminus, a major component of the chemokine-GPCR interface. Replacement of the X residue with the most frequent amino acid at this position (P10Q) had an intermediate effect between WT and P10del in each assay, with ACKR3 having a higher tolerance for this mutation. This work shows that the X residue helps to position the CXCL12 N terminus for optimal docking into the orthosteric pocket of CXCR4 and suggests that the CC/CXC motif contributes directly to receptor selectivity by orienting the chemokine N terminus in a subfamily-specific direction.

Heterologous regulation of CXCR4 lysosomal trafficking.

G protein-coupled receptor (GPCR) signaling is regulated by members of the protein kinase C (PKC) and GPCR kinase (GRK) families, although the relative contribution of each to GPCR function varies among specific GPCRs. The CXC motif receptor 4 (CXCR4) is a member of the GPCR superfamily that binds the CXC motif chemokine ligand 12 (CXCL12), initiating signaling that is subsequently terminated in part by internalization and lysosomal degradation of CXCR4. The purpose of this study is to define the relative contribution of PKC and GRK to CXCR4 signaling attenuation by studying their effects on CXCR4 lysosomal trafficking and degradation. Our results demonstrate that direct activation of PKC via the phorbol ester phorbol 12-myristate 13-acetate (PMA) mimics CXCL12-mediated desensitization, internalization, ubiquitination, and lysosomal trafficking of CXCR4. In agreement, heterologous activation of PKC by stimulating the chemokine receptor CXCR5 with its ligand, CXCL13, also mimics CXCL12-mediated desensitization, internalization, ubiquitination, and lysosomal degradation of CXCR4. Similar to CXCL12, PMA promotes PKC-dependent phosphorylation of serine residues within CXCR4 C-tail that are required for binding and ubiquitination by the E3 ubiquitin ligase AIP4 (atrophin-interacting protein 4). However, inhibition of PKC activity does not alter CXCL12-mediated ubiquitination and degradation of CXCR4, suggesting that other kinases are also required. Accordingly, siRNA-mediated depletion of GRK6 results in decreased degradation and ubiquitination of CXCR4. Overall, these results suggest that PKC and GRK6 contribute to unique aspects of CXCR4 phosphorylation and lysosomal degradation to ensure proper signal propagation and termination.

Endocytosis is required for CXC chemokine receptor type 4 (CXCR4)-mediated Akt activation and antiapoptotic signaling.

Signaling activated by binding of the CXC motif chemokine ligand 12 (CXCL12) to its cognate G protein-coupled receptor (GPCR), chemokine CXC motif receptor 4 (CXCR4), is linked to metastatic disease. However, the mechanisms governing CXCR4 signaling remain poorly understood. Here, we show that endocytosis and early endosome antigen 1 (EEA1), which is part of the endosome fusion machinery, are required for CXCL12-mediated AKT Ser/Thr kinase (Akt) signaling selective for certain Akt substrates. Pharmacological inhibition of endocytosis partially attenuated CXCL12-induced phosphorylation of Akt, but not phosphorylation of ERK-1/2. Similarly, phosphorylation of Akt, but not ERK-1/2, stimulated by CXCL13, the cognate ligand for the chemokine receptor CXCR5, was also attenuated by inhibited endocytosis. Furthermore, siRNA-mediated depletion of the Rab5-effector EEA1, but not of adaptor protein, phosphotyrosine-interacting with PH domain and leucine zipper 1 (APPL1), partially attenuated Akt, but not ERK-1/2, phosphorylation promoted by CXCR4. Attenuation of Akt phosphorylation through inhibition of endocytosis or EEA1 depletion was associated with reduced signaling to Akt substrate forkhead box O1/3a but not the Akt substrates TSC complex subunit 2 or glycogen synthase kinase 3ß. This suggested that endocytosis and endosomes govern discrete aspects of CXCR4- or CXCR5-mediated Akt signaling. Consistent with this hypothesis, depletion of EEA1 reduced the ability of CXCL12 to attenuate apoptosis in suspended, but not adherent, HeLa cells. Our results suggest a mechanism whereby compartmentalized chemokine-mediated Akt signaling from endosomes suppresses the cancer-related process known as anoikis. Targeting this signaling pathway may help inhibit metastatic cancer involving receptors such as CXCR4.