br Crystal structure of c
Crystal structure of c-FMS and binding pattern of CSF-1 and IL-34 c-FMS is a 972 amino acids polypeptides containing transmembrane glycoprotein . It contains all the necessary domains required for tyrosine kinase activity, i.e. 512 amino LY-411575 N-terminal extracellular segment, hydrophobic 25 amino acid membrane spanning region, a 435 amino acid intracellular domain. Various protein tyrosine kinases contain 60–100 residues with different sequence . The overall structure of c-FMS-PTK, c-KIT  and FLT-3  closely resemble with each other i.e a typical bi-lobal PTK folds and JM (Juxtamembrane domain) domain PTK domain contains two lobes: N and C-terminal lobe. The N-terminal lobe of cFMS-PTK contains- five stranded anti-parallel β-sheet (β1-β5) and a single α-helix. C-terminal lobe contains-seven α-helices (αD, αE, αEF, αF-αI) and two β strands (β6 and β7) and activation loop located between β7 and αEF (residues 796–825). (Fig. 2.1 A) . Its auto-inhibited structure contain N-terminus packed between residues of the glycine-rich loop, the activation loop and the αC helix. Both its N and C termini wrapping around the αC helix and the JM domain adopts a twisted hairpin conformation (Fig. 2.1B). The exclusive and unphosphorylated tyrosine, Tyr 809 points towards the active site surrounded by hydrophobic residues Pro818, Ile803 forming hydrogen bonds with Asp778 and Arg801. (Fig. 2.1C). The JM domain can be divided into three subsections-JM-B (buried region), JM-S (switch motif) and JM-Z (zipper region) (Fig. 2.2 A). The JM domain is responsible for the autoinhibitory mechanism, containing tyrosine residues- Tyr546, Tyr561 and Tyr571 (Fig. 2.2B). The first seven residues of cFMS JM-B make direct connection with αC helix, the catalytic and activation loop (corresponding to residues Tyr546-Ile553). JM-S is a switch motif having hairpin like conformation and JM-Z, having ten residue section (Asp565-Lys574), also known as zipper or linker region . CSF-1 is a 554 amino acid protein contain three components- N-terminal 32 amino acid signal pepetide, a 149 residue growth factor domain, a 24 residue transmembrane region and a 35 amino acid cytoplasmic tail. The CSF-1 covalently attached to D2 and D3 domains of receptor which causes the homodimerisation through D4 and D5 domains and leads to signal transduction pathway [25,26]. IL-34 is a 241 amino acid protein in humans having 26% sequence similarity with CSF-1. It has two isoforms with the difference of insertion position of glutamine. IL-34 is a homodimeric protein formed by four α-helix and disulfide bond [27,28]. As seen in the Fig. 2.3 , the mode of binding of IL-34 and CSF-1 with CSF-1R is same in terms of the domains involved. IL-34 undergo the rotation between D2 and D3 domains and attain an elongated structure. The IL-34 interaction with D2 domain involve α-B and α-C helices and α3 loop. In case of interaction with D3 domain α-A, α-C and α4 helices. However, the interaction sites are more covered in case of IL-34 as compared to CSF-1 . The CSF-1R is composed of five immunoglobulin-like domains- D1,D2,D3,D4 and D5. Because of the similar binding mode, IL-34 fights with CSF-1 for receptor site . The combined effect of both cytokines is different as compared to the single dose effect of CSF-1 and IL-34. The attachment of two ligands with CSF-1R is due to flexibility between D2 and D3 [4,7]. In comparing both free and bound forms, no major difference was found in the crystalline structure of IL-34  (Fig. 2.4 ).
Mechanism of signal transduction of CSF-1 and IL-34 through CSF-1R/ c-FMS Receptor tyrosine kinases (RTKs) are essential components of signal transduction pathways. These transmembrane receptors, which bind polypeptide ligands mainly growth factors, play key roles in processes such as cellular growth, differentiation, metabolism and motility. RTKs are active during embryonic development and adult homeostasis . Signal transduction through CSF-1 ligand receptor complex leads to the differentiation and proliferation of cells of the monocyte/macrophage lineage . Over expression of CSF-1 has been implicated in a number of disease states such as cancer and inflammation. CSF-1 released by osteoblasts, stimulates the proliferation of osteoclasts in combination with receptor activator of nuclear factor κB (RANK) (Fig. 2.5 ). It involves at least three parallel pathways, i.e. Src pathway, MEK1/extracellular signal-regulated kinase (ERK) pathway, and the c-MYC pathway . On the binding of CSF-1 with its receptor (CSF-1R), it undergoes differentiation and proliferation of osteoclasts with the proliferation of various cytokines in the sequence from Src to c-myc. Phosphorylated c-myc enters the nucleus and eventually leading to expression of an essential regulator of osteoclasts differentiation AP-1. Further PI3K-AKT and PKC pathways are found to be involved in M-CSF stimulated NF-κB activation. PKCs are involved in different cell responses- cell growth, survival, differentiation and development . Finally, they induce NF-κB transcriptional activity which is a key regulator of genes Bc-XL and IκB. NF-κB activation is important in M-CSF-induced monocyte survival . In addition to its role in mononuclear phagocyte survival, the transcriptional factor NF-κB regulates numerous genes that play important roles in cellular signalling, stress responses, cell growth, survival, differentiation and inflammation .