G-protein coupled receptors (GPCRs) are integral proteins present embedded in cell membrane. It contains 7-transmembrane sequential α-helical regions with extracellular binding domain. GPCR plays an important role in cell signaling cascade. It involves in signal transduction via interacting with wide range of extracellular ligands (hormones, neurotransmitters etc.) and transduce their signals to G-proteins. G-proteins are heterotrimeric structures with Gα, Gẞ and Gγ subunits which further transduce intracellular signaling. Upon binding of the appropriate ligand, GPCR undergoes conformational changes and activates G-protein. In the absence of a signal, Gα subunit present bound to GDP and G-protein is in its inactive form. Once the receptor is activated, G-protein binds to the GPCR and GPCR induces a conformational change in Gα causing replacement of GDP by GTP. Binding of GTP induces further conformational changes in G-protein causing detachment of Gα from Gẞγ. The GTP-Gα complex then activates the secondary messengers such as cyclic adenosine monophosphate (cAMP), cGMP, inositol-1, 4, 5-triphosphate (IP3), phospholipase C and diacylglycerol (DAG) initiating downstream intracellular signaling cascades that involve in normal cell functions. After the completion of transduction of primary messengers into secondary messengers, eventually, Gα comes to its inactive form by hydrolysing GTP to GDP. Then it re-forms the Gαẞγ complex; the inactive form of G-protein (Figure 1) (Tuteja, 2009).

Figure 1: Model for signal transduction by activation/inactivation of heterotrimeric G-proteins through GPCR. In the inactive state heterotrimeric G-protein subunits are associated with each other. In the inactive state GDP is present bound to Gα. Once the GPCR got stimulated with the binding of an extracellular ligand, it undergoes conformational changes and activates the inactive G-protein complex causing dissociation of Gα from Gẞγ. In the active state, GDP is replaced by GTP. Then the two separated Gα-GTP and Gẞγ complexes bind with their own receptors E1 & E2 for further signal transduction. Once the signal transduction is completed, GDP hydrolyses back into GDP and Pi and inactivate the G-protein complex by binding with Gẞγ. In this way activation and inactivation, cycle is completed.

Chemokine receptors belong to GPCR superfamily and are divided into 4 main chemokine receptor groups as CXC, CX3C, CC and C respectively. This classification depends on the position of highly conserved cysteins in the amino acid sequence and according to the chemokines with which they interact. Chemokines are small chemoattractant peptides which are a subtype of cytokines that involve in chemotaxis. They are associated with immune cell recruitment, migration and most importantly homeostasis (Finn and Murdoch, 2000). Chemokines interact with heterotrimeric G-proteins to bring about their actions on target cells with the aid of GPCRs known as chemokine GPCRs (Figure 2). CXC chemokine receptor (CXCR) is a prototype receptor for chemokine GPCRs and contains 7 CXCRs named CXCR1-7 (Ransohoff, 2009).

Figure 2: Schematic summary of general mechanism of chemokine-mediated GPCR signaling and production of secondary messengers such as cyclic AMP, cyclic GMP, inositol triphosphate (IP3), diacylglycerol (DAG) and calcium which further generate intracellular signal transduction (McNeil and Patel, 2013).

To date there are about 22 chemokine receptors and 50 chemokine ligands are present (Pease and Solari, 2015). Chemokine receptors are predominately expressed in the membranes of leukocytes (Figure 3) (Lodowski and Palczewski, 2009). Chemokine function plays an important role in leukocyte trafficking, cardiogenesis, neural differentiation, hematopoiesis and organogenesis (Locati, 1999). Simply, chemokine GPCR mediated coordination of leukocyte activities such as movement and positioning of leukocytes help development, maintenance and proper functioning of immune system. However, the aberrant expression of chemokine receptors and signaling contribute for assemblage of pathologies which cause improper leukocyte recruitment leading to diseases such as inflammatory, infectious, autoimmune, neurodegenerative diseases and cancers (Bennett, Fox and Signoret, 2011). Therefore, chemokine GPCRs have become an important target in therapeutic intervention and disease therapy (Doijen et al, 2017).

Leukocyte expression and specific ligand-chemokine receptor interactions

Figure 3: Leukocyte expression and specific ligand-chemokine receptor interactions (Bonecci et al, 2015).

Role of chemokine GPCRs in human immunodeficiency virus (HIV)

The chemokine GPCR, CXCR4 and CXCR3 transduce signals of their chemokine ligands CXCL12 and CXCL10 respectively. The over-expression of CXCR4 and CXCR3 have been proven to be associated with HIV progression. CXCR4 mediates HIV viral entry while CXCR3 involves in recruitment of T-cells into local HIV infected lymph nodes (Jiang, Shang and Wang, 2017; Lodowski and Palczewski, 2009; Fujii et al, 2007). T-cell trophic HIV-1 which is the etiological agent of acquired immunodeficiency syndrome (AIDS), uses CD4 and CXCR4 to assist the envelope mediated cell fusion and entry (Figure 4) (Finn and Murdoch, 2000).

HIV life cycle

Figure 4: HIV life cycle. The entry begins with binding of the viral envelope to CD4 via envelope glycoprotein spikes present on the surface of HIV envelope. This induces conformational changes in HIV gp120 subunit exposing chemokine receptor binding site on gp120. This facilitate the binding of gp120 with CXCR4 present on the surface of the target cell. This binding of gp120 with CXCR causes further conformational changes exposing gp41 subunit. The gp41 inserts the coiled helical structures and initiate viral and target cell membrane fusion allowing the entry of viral core into the target cell. Eventually, viral RNA replicates and newly form HIV particles will be bud-off from the host cell and released into the circulation (Adapted from Jef, 2014).

Even in the absence of CD4, CXCR4 has the ability to detect the T-trophic-virus binding site. Hence CXCR4 permits HIV entry even into cells that do not express CD4. This shows that HIV uses CD4 to present the virus to the chemokine GPCR (Finn and Murdoch, 2000). The binding of natural ligand CCL12 to CXCR4 activates actin-related signaling pathway. Actin usually forms a dense network acting as a barrier to the pathogen entry. Hence, HIV gp120 competes with the natural ligand CXCL12 for CXCR4 binding and inhibits the binding of CXCL12 (Figure 5) (Jiang, Shang and Wang, 2017).

Figure 5:possible mechanisms of chemokine-induced actin activation in the promotion of HIV infection. HIV gp120 binds to CXCR4 inhibiting the interaction of CXCL12. Two major actin related pathways are activated. They are, LIMK1-coflin and WAVE2-Arp2/3 pathways which induces actin polymerization and de-polymerization resulting in cytoskeletal rearrangement. This enhances the HIV fusion, entry, nuclear integration, release and transmission (Jiang, Shang and Wang, 2017).

The T-lymphocyte depletion by gp120-CXCR4 binding accelerates the HIV-1 progression. Therefore, multiple studies have been focused on therapeutic intervention by targeting the gp120-CXCR4 interaction. This approach may useful in preventing the late-stage of infection (Chan et al, 2000). To date there are several CXCR4(X4) antagonists involve in HIV-1 such as T22, T140, FC131 and KRH-3955 (Ohasi and Tamamura, 2016; Hamatake et al, 2009; Fujii et al, 1999). They competitively bind to X4 receptor preventing ligand binding to CXCR4. Thereby, inhibits the HIV-1 binding to the target cells and further progression (Figure 6). However, majority of these antagonists are at preclinical stage (Table 1) and some are suspended (AMD070, ALX40-AC) (Murakami and Yamamoto, 2010).

Therapeutic opportunities for inhibition of HIV-1 entry

Figure 6: Therapeutic opportunities for inhibition of HIV-1 entry (Adapted from Davis et al, 2009)

Table 1: Effects of X4 HIV-1 antagonist (Adapted from Hamatake et al, 2009)

The novel CXCR4 antagonist KRH-3955 is an orally bioavailable and extremely potent inhibitor of HIV-1.KRH-3955 potently and selectively inhibited the X4 HIV-1 binding and replication. Inhibits the binding of natural ligand by competitively binding to X4 and prevents the binding of HIV-1 to CXCR4.Figure 7: Inhibitory effects of KRH-3955 on chemokine binding to CXCR4.KRH-3955 is a promising antagonist in X4 HIV-1 binding and an HIV-1 anti-viral agent.

Role of Chemokine GPCRs in Glioblastoma Multiforme (GBM)

GBM is the most common and aggressive type of cancer that occurs in the brain. GBM tumors are begin in astrocytes which involve in neuronal nourishment and repair. Elevated levels of CXCR4 and CXCR7 are found to be associated with cancer stem-like cells (CSC) in GBM (Figure 8). CXCL12 is the common chemokine ligand for both CXCR4 and CXCR7 activation. The altered expression of these receptors enhance tumor survival, growth, proliferation and angiogenesis (Figure 9) (Bajetto et al, 2014; Foltz et al, 2013).

CXCL12/CXCR4-CXCR7 system in the GBM CSC niche

Figure 8: CXCL12/CXCR4-CXCR7 system in the GBM CSC niche. This is the tumor microenvironment which contains tumor cells, CSCs, blood vessels etc. These cells secrete CXCL12 and generates intracellular pathways such as protein kinase B/ mitogen-activated protein kinase (Akt/MAPK) signaling which contribute for CSC self-renewal, survival and migration (Bajetto et al, 2014).

chematic diagram of proposed CXCR4-CXCR7

Figure 9:Schematic diagram of proposed CXCR4-CXCR7 crosstalk affecting major signaling pathways related to cell survival, proliferation and migration.Upon CXCL12 binding to CXCR4 and CXCR7, receptor homo or hetero dimerization occurs. Ligand binding causes conformational changes in CXCR4/CXCR7 receptors and activates G-proteins which induce multiple signaling pathways. Gα inhibits cAMP formation through modulation of adenylyl cyclase (AC) and Gγ activates phospholipase-C inducing DAG/IP3 pathways which involve in Ca2+ release. Thereby regulates cell survival, proliferation and chemotaxis. Gα activates nuclear factor-κB (NFκB) and Ca2+ tyrosine kinase pathways such as Janus kinase/signal transducer and activator transcription protein (JAK/STAT) and PI3K/Akt pathways leading cell survival and proliferation. Gẞγ activates Ras which induces the activation of extracellular regulated kinase ½ mitogen-activated protein kinase (ERK1/2 MAPK) pathway resulting in changes in genes expression and cell cycle progression. Gẞγ also activates PI3K and modulate CXCL12-dependent chemotaxis. CXCL12 also involves in endocytosis of CXCR4 with the aid of ẞ-arrestin. Interaction of CXCR4-ẞ-arrestin further activate further downstream intracellular signaling pathways which may induce cell migration. Expression of CXCR4/CXCR7 promotes GBM growth, survival, proliferation, migration and metastasis (Bajetto et al, 2014).

Table 2:  CXCR4 and CXCR7 as therapeutic targets in GBM (Adapted from Bain et al, 2015; Liu et al, 2015).

miR-663 suppresses oncogenic function of CXCR4 in GBMSuppressed the CXCR4  expression andmiR-663 in combination with CXCR4 antagonist (AMD3100) enhanced the tumor-suppressive effects.

Elevated oncogenic microRNA levels modulate the gene expression and increases the expression of CXCR4. miR-633 suppresses elevated microRNA levels and regulate the expression of CXCR4 while AMD3100 blocks the CXCR4 binding with ligand. Figure 10:Upregulated miR-663 reduces the proliferation and invasion of GBM cells promoted by CXCR4 overexpression (Bain et al, 2015).miR-633 is a potential agent in treating GBM and in combinatorial therapeutic approach.
Targeting CXCR7 inhibits glioma cell proliferation & mobilitySuppression of CXCR7 expression by gene silencing RNA (siRNA) and CXCR7 specific antagonist CCX777 inhibits glioma cell proliferation and invasion. Suppression of CXCR7 expression by siRNA reduces ERK ½ phosphorylation. CCX777 blocks the CXCR7. Thereby, inhibits glioma cell proliferation and invasion. Figure 11: Knockdown of CXCR7 by siRNA (Liu et al, 2015).Targeting CXCR7 may provide novel opportunities for improving glioma therapy.

In summary, chemokine GPCRs diversely involve in disease progression. Therefore, they are plausible drug targets in therapeutic intervention which mainly involves in blocking the chemokine GPCRs via small molecule antagonists and monoclonal antibodies (Pease and Solari, 2015). The prototype receptor CXCR has also become an important druggable target molecule as it involves in many disease states including HIV, autoimmune, several immunodeficiency disorders and 23 types of cancers. Therefore by understanding the receptor structure, interactions and signaling pathways that involve in disease progression; safe, effective and more specific drug opportunities can be developed.  


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