skip to main content


Title: Differential activity and selectivity of N-terminal modified CXCL12 chemokines at the CXCR4 and ACKR3 receptors
Abstract

Chemokines play critical roles in numerous physiologic and pathologic processes through their action on seven-transmembrane (TM) receptors. The N-terminal domain of chemokines, which is a key determinant of signaling via its binding within a pocket formed by receptors’ TM helices, can be the target of proteolytic processing. An illustrative case of this regulatory mechanism is the natural processing of CXCL12 that generates chemokine variants lacking the first two N-terminal residues. Whereas such truncated variants behave as antagonists of CXCR4, the canonical G protein-coupled receptor of CXCL12, they are agonists of the atypical chemokine receptor 3 (ACKR3/CXCR7), suggesting the implication of different structural determinants in the complexes formed between CXCL12 and its two receptors. Recent analyses have suggested that the CXCL12 N-terminus first engages the TM helices of ACKR3 followed by the receptor N-terminus wrapping around the chemokine core. Here we investigated the first stage of ACKR3-CXCL12 interactions by comparing the activity of substituted or N-terminally truncated variants of CXCL12 toward CXCR4 and ACKR3. We showed that modification of the first two N-terminal residues of the chemokine (K1R or P2G) does not alter the ability of CXCL12 to activate ACKR3. Our results also identified the K1R variant as a G protein-biased agonist of CXCR4. Comparative molecular dynamics simulations of the complexes formed by ACKR3 either with CXCL12 or with the P2G variant identified interactions between the N-terminal 2–4 residues of CXCL12 and a pocket formed by receptor's TM helices 2, 6, and 7 as critical determinants for ACKR3 activation.

 
more » « less
NSF-PAR ID:
10392479
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ; ;
Publisher / Repository:
Oxford University Press
Date Published:
Journal Name:
Journal of Leukocyte Biology
Volume:
107
Issue:
6
ISSN:
0741-5400
Page Range / eLocation ID:
p. 1123-1135
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Homodimeric class 1 cytokine receptors include the erythropoietin (EPOR), thrombopoietin (TPOR), granulocyte colony-stimulating factor 3 (CSF3R), growth hormone (GHR), and prolactin receptors (PRLR). These cell-surface single-pass transmembrane (TM) glycoproteins regulate cell growth, proliferation, and differentiation and induce oncogenesis. An active TM signaling complex consists of a receptor homodimer, one or two ligands bound to the receptor extracellular domains and two molecules of Janus Kinase 2 (JAK2) constitutively associated with the receptor intracellular domains. Although crystal structures of soluble extracellular domains with ligands have been obtained for all the receptors except TPOR, little is known about the structure and dynamics of the complete TM complexes that activate the downstream JAK-STAT signaling pathway. Three-dimensional models of five human receptor complexes with cytokines and JAK2 were generated here using AlphaFold Multimer. Given the large size of the complexes (from 3220 to 4074 residues), the modeling required a stepwise assembly from smaller parts with selection and validation of the models through comparisons with published experimental data. The modeling of active and inactive complexes supports a general activation mechanism that involves ligand binding to a monomeric receptor followed by receptor dimerization and rotational movement of the receptor TM α-helices causing proximity, dimerization, and activation of associated JAK2 subunits. The binding mode of two eltrombopag molecules to TM α-helices of the active TPOR dimer was proposed. The models also help elucidating the molecular basis of oncogenic mutations that may involve a non-canonical activation route. Models equilibrated in explicit lipids of the plasma membrane are publicly available. 
    more » « less
  2. CC chemokine receptor 2 (CCR2) is a part of the chemokine receptor family, an important class of therapeutic targets. These class A G-protein coupled receptors (GPCRs) are involved in mammalian signaling pathways and control cell migration toward endogenous CC chemokine ligands, named for the adjacent cysteine motif on their N terminus. Chemokine receptors and their associated ligands are involved in a wide range of diseases and thus have become important drug targets. CCR2, in particular, promotes the metastasis of cancer cells and is also implicated in autoimmunity-driven type-1 diabetes, diabetic nephropathy, multiple sclerosis, asthma, atherosclerosis, neuropathic pain, and rheumatoid arthritis. Although promising, CCR2 antagonists have been largely unsuccessful to date. Here, we investigate the effect of an orthosteric and an allosteric antagonist on CCR2 dynamics by coupling long-timescale molecular dynamics simulations with Markov-state model theory. We find that the antagonists shift CCR2 into several stable inactive conformations that are distinct from the crystal structure conformation and disrupt a continuous internal water and sodium ion pathway, preventing transitions to an active-like state. Several metastable conformations present a cryptic drug-binding pocket near the allosteric site that may be amenable to targeting with small molecules. Without antagonists, the apo dynamics reveal intermediate conformations along the activation pathway that provide insight into the basal dynamics of CCR2 and may also be useful for future drug design.

     
    more » « less
  3. Abstract

    Fibroblasts are an abundant cell type in tumor microenvironments. Activated fibroblasts, known as carcinoma‐associated fibroblasts (CAFs), interact with cancer cells through biochemical signaling and render cancer cells proliferative, invasive, and resistant to therapeutics. Targeting CAFs–cancer cells interactions offers a strategy to block cancer progression. 2D and 3D co‐cultures of human mammary fibroblasts and triple negative breast cancer (TNBC) cells are used to investigate the impact of heterotypic cellular interactions on the proliferation of matrix invasion of TNBC cells. The results show that fibroblasts secreting a chemokine, CXCL12, significantly enhance proliferation of TNBC cells expressing the chemokine receptor, CXCR4. Disrupting this interaction with a receptor antagonist normalizes cancer cell proliferation to that of a co‐culture model lacking this signaling. When co‐culture spheroids are embedded in collagen, fibroblasts producing CXCL12 promote collagen invasion of TNBC cells. Although co‐cultures containing normal fibroblasts also lead to TNBC cell spreading into the matrix, a morphological analysis of cells and inhibition of chemokine‐receptor signaling shows that this spreading is due to the incompatibility of fibroblasts and cancer cells leading to the segregation of the two cell types from the spheroid.

     
    more » « less
  4. Abstract

    Neuropeptide Y (NPY) is highly abundant in the brain and involved in various physiological processes related to food intake and anxiety, as well as human diseases such as obesity and cancer. However, the molecular details of the interactions between NPY and its receptors are poorly understood. Here, we report a cryo-electron microscopy structure of the NPY-bound neuropeptide Y1 receptor (Y1R) in complex with Gi1protein. The NPY C-terminal segment forming the extended conformation binds deep into the Y1R transmembrane core, where the amidated C-terminal residue Y36 of NPY is located at the base of the ligand-binding pocket. Furthermore, the helical region and two N-terminal residues of NPY interact with Y1R extracellular loops, contributing to the high affinity of NPY for Y1R. The structural analysis of NPY-bound Y1R and mutagenesis studies provide molecular insights into the activation mechanism of Y1R upon NPY binding.

     
    more » « less
  5. Abstract

    Trafficking of leukocytes and their local activity profile are of pivotal importance for many (patho)physiological processes. Fittingly, microenvironments are complex by nature, with multiple mediators originating from diverse cell types and playing roles in an intimately regulated manner. To dissect aspects of this complexity, effectors are initially identified and structurally characterized, thus prompting familial classification and establishing foci of research activity. In this regard, chemokines present themselves as role models to illustrate the diversification and fine-tuning of inflammatory processes. This in turn discloses the interplay among chemokines, their cell receptors and cognate glycosaminoglycans, as well as their capacity to engage in new molecular interactions that form hetero-oligomers between themselves and other classes of effector molecules. The growing realization of versatility of adhesion/growth-regulatory galectins that bind to glycans and proteins and their presence at sites of inflammation led to testing the hypothesis that chemokines and galectins can interact with each other by protein–protein interactions. In this review, we present some background on chemokines and galectins, as well as experimental validation of this chemokine–galectin heterodimer concept exemplified with CXCL12 and galectin-3 as proof-of-principle, as well as sketch out some emerging perspectives in this arena.

     
    more » « less