10/29/2013
D1398G Variant of HGF Receptor – Disclosure A Potential Biomarker of Severe ILD in African American Scleroderma Patients Nothing to disclose
Galina S. Bogatkevich Medical University of South Carolina
HGF Receptor Role of C-MET in Fibrotic Remodeling Cellular Mesenchymal-Epithelial Transition Factor C-MET Tissue Injury
• Embryogenesis and development Epithelial / Endothelial HGF/MET Myofibroblast Apoptosis • Primary function in the adult is tissue repair Cell Survival /Proliferation • Promotes normal tissue regeneration and prevents fibrotic Epithelial / Endothelial Fibroblast Proliferation remodeling in the lung, skin, kidney, liver, and heart Cell Death Collagen, CTGF Myofibroblast Activation • Expressed mainly on epithelial cells and myofibroblasts • Very low expression in normal fibroblasts Fibrotic Remodeling
C-MET C-MET Transcription Cellular Mesenchymal-Epithelial Transition Factor HGF Receptor
1 1 308 • 1408 amino acids Tyrosine Kinase 290 (308) Several 5’ alternative splicing are known • Synthesized as 170 kDa single chain precursor • Glycosylated mature form of 307 289 (307) 50 kDa α-chain and 140 kDa β-chain - 18 amino acids (exon 10) = 1390 aa linked by disulphide bridges • α-chain – ligand-binding pocket • β-chain – extracellular, transmembrane,
1408 Tyrosine kinase, C-terminal 1390 (1408)
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Human c-met Gene SNPs, Germline, and Somatic Mutations
Identified in Human MET Gene
Exon 21
Exon 2
TK Exons 16 - 19
Y1248C/D/H
K1262R
Y1246N/H
M1268T
H1112R/L/Y
G1137V
Y1230D/C/H/D Y1235D
V1206L L1213V
H1124D
T1010I V1110I T1191I
M1149T V1238I Insertion P1009S
• Chromosome 7q21-q31 1 Exon 2 TK Exons 16 - 19 Exon 21 1408
E14
• 120 kb D1398G
R988C
L238SL238S
N375SN375S
R143QR143Q
T309PT309P
I316M I316M A320V • 21 exons (8 kb) separated by 20 introns A320V • Exon 21 includes C-terminus + large 3’UTR • Coding region 4 kb
D1398G identified in African American SSc-ILD patients and SNPs affecting African American populations are shown in red.
Germline, and Somatic Mutations Identified in Human MET Gene
• All known mutations in human Met gene are associated with cancer • 10 known germinal mutations are responsible for hereditary papillary renal carcinoma • Somatic missense mutations are associated with sporadic papillary renal carcinoma, hepatocellular carcinoma, gastric carcinoma, glioma, head and neck squamous cell carcinomas
Defective HGF Signaling in Lung Fibroblasts
HGF - + - + - + - + - + - + - + - + - + HGF - + - + - + - + - + - + - + - + - + CTGF Ph-c-Met
c-Met Collagen Type I
β-actin b-actin 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 Caucasian African-American Cell Lines Caucasian African-American SSc Cell Lines
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C-Met Genetic Analysis C-Met Genetic Analysis
MET1 TGGTGCAGAGGAGCAATG 1TEM TTGCTTTGTGCGAACACC
MET2 CGGTCCAAAGGGAAACTC 2TEM TCATGGCAGGACCAACTG
MET3 CTGGTGCCACGACAAATG 3TEM GGGACCGTGCATAAAACG
MET4 CCCCCTGAAAACCAAAGC 4TEM TGGGCAGTATTCGGGTTG Glycine MET5 CTGCGAAGTGAAGGGTCTC 5TEM GACCAAGCCTCTGGTTCTG Aspartic Acid Alanine
• Aspartic acid carries a hydrophilic group with strong negative charge located on the outer surface of protein
• It has been reported to be essential in several proteins for ligand recognition and phosphorylation
• Glycine is a neutral amino acid with reduced reactivity
Glycine Aspartic Acid
Is D1398G Responsible for Defective C-MET Cellular Mesenchymal-Epithelial Transition Factor HGF Signaling in Lung Fibroblasts? HGF Receptor
Distinct from other RTKs due to: • Transfect Lung fibroblasts with 1. Unique multi-substrate docking site Met WT and D1398G • Incubate cells with and without HGF 2. Signaling through an adaptor protein • Evaluate c-Met phosphorylation, unique to c-Met, Grb2-associated binder (Gab)-1 • Collagen, and CTGF expression Y1252 Y1253 Y1367, Y1374 Docking sites D1398G
D1398G MET as a Potential Biomarker of MET Signaling Pathways in Lung Fibroblasts Severe SSc-ILD
Prognosis for pulmonary outcomes is worse and the mortality rate among HGF African Americans with SSc is twice as high when compared with whites
African Whites Americans SSc-ILD Severity : ? Current SSc-ILD patients (LF isolated Y1252, Y1253 c-MET SNPs from BAL samples): Gab1 Y1367, Y1374 D1398G HRCT, PFT, Mahler Dyspnea Index Grb2 D1398G c-MET SOS LF from patients with terminal stage of Ras Collagen SSc-ILD: MMP MAPK NF-κB CTGF Increased CTGF SSc-lLD Severity Age at onset and SSc-ILD duration
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Acknowledgments
P60 AR062755-01 Project 1 of MCRC for Rheumatic Diseases in African Americans
Richard M. Silver Ilia Atanelishvili Tanjina Akter Yuichiro Shirai C. Beth Singleton
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