CXCR4 Introduction• G protein coupled receptor (GPCR)• Activated by SDF-1, a chemokine• Thought to be important to two major diseases• HIV- Acts as co-receptor for M Tropic• Possible cure for M Tropic infections• Cancer- Expressed more in breast and other cancers• Aids in metastasis of cancer to lungs and bones•Stop CXCR4 from working, metastasis would stop to lungs and bones
Project
• The purpose of the project was to mutate the CXCR4 Constitutively Active Mutant (CAM) receptor, do experiments in order to determine whether or not the receptor is active given the random mutations, and to find out exactly where and what the mutations are.
• Experiments:
1) Filter Lift Assay 6) PCR
2) His Growth Assay 7) Western Blot
3) Plasmid Manipulation 8) Sequencing
4) Restriction Digest 9) Mutagenesis
5) Fluorescent Lac Z 10) 3-D Modeling
Filter Lift Assay
• Quickest and easiest method to mass screen for receptor activity• Yeast is grown on an agar plate with a grid in order to identify the
colonies. The colonies are the lifted with a piece of filter paper that is pre-soaked in X Gal and Z Buffer.
• The yeast are permeabilised and then incubated over 2-3 hours and the active colonies turned blue.
• Yeast with an active receptor synthesize B-Galactosidase via FUS-1 activation. X Gal is a substrate for B-Gal. Complete X Gal is colorless; cleaved X Gal is blue.
X X X
X X X X X X
X X X X X X X X X
X X X X X X
X X X
His Growth Assay
• Yeast is grown in a medium that lacks a necessary amino acid- Histidine.
• This test is used to confirm the filter lift. If the yeast grows then it is active.
His Growth Assay of SingleMutants
0 10 20 300.00
0.25
0.50
0.75wtn119sM63L1
V82G1V82G2
V82G3
F248I1
F248I2
F248I3
Time (Hours)
OD
600
Plasmid Manipulation
• The plasmid from the revert mutants is extracted by minipreps. • The plasmid is then transformed into Nova Blue competent cells• The DNA is extracted from the competent cells and used for
downstream experiments and sequencing.
Selection Procedure• Different mediums select different plasmids i.e.:
CP4258 –Leu
CP1584 –Trp • Different antibiotics select different plasmids in bacteria i.e.:
Cp4258 & CP1584 are grown in Ampicillin
Restriction Digest
• Cuts plasmid and shows whether or not CXCR4 is present
• The plasmid is cut with restriction enzymes ( XbaI and NcoI)
NcoI- CCATGG
XbaI- TCTAGA
PCR
• A polymerase is added to DNA and replicates the desired DNA.
• The primers have complementary sequences that line up with the desired segment of the DNA.
• Taq polymerase adds nucleotides to make up the DNA.
• Used to make sure CXCR4 is present.
Fluorescent Lac Z
• Same principle as filter lift.
• FDG is used as substrate in place of X Gal.
• When cleaved, FDG fluoresces.
Fluorescent Lac Z Assay of Single Mutants
wt N119S M63L1 M63L2 M63L3 V82G1 V82G2 V82G3 F248I10
25000
50000
75000
Mutants
Flu
ore
scen
ce
Western Blot
• Shows if protein is present in sample.
• 9E10 is the antibody that binds to Myc tagged CXCR4.
• Used to see the size of protein present.
• Results came out blank.
Sequencing
• DNA is sent to be sequenced.• Results where mutations, if any, are present.• Mutations that we found: Mutation Rate:
Revert 2-13: V82G 1/1089
Revert 2-16: V82G 1/1089
Revert 2-21: M63L, S312P 2/1089
Revert 2-26: S123I, E288G 4/1089
Revert 2-32: F29S, V59A, F248I, V320E 4/1089
Revert 2-49: F248I 2/1089
Overall Mutation Rate: 14/6532= .21%
Mutagenesis
• Site directed mutagenesis of CXCR4 CAM• Separates multiple mutations• Mutate into pcDNA3.1zeo(+) for transfection into Mammalian Cells• Mutations:
M63L
V82G
F248I
E288G
3-D Modeling
• Displayed mutations on CXCR4• Saw possible interactions with other residues• Possible important hydrophobic interactions:
S123: W252, N119
L244: L127, S123, Y76, I126
L246: H228, L301
F248: Y256, L253, I215, L127
PLATE
1PLATE
2PLATE
3PLATE
4PLATE
5PLATE
6PLATE
7PLATE
8PLATE
9 TOTAL
Filter Lift 51 44 47 45 45 48 49 47 48 424
His Growth 0 36 47 38 44 41 0 40 0 246
Yeast Miniprep 0 33 43 21 43 31 0 37 0 208
Bact Transformation 0 6 5 0 0 11
Bact MinipreP 0 6 5 0 0 11
Sal 1 and bact transf. Miniprep 0 6 5 0 0 11
Yeast TRAnsformation (lac Z) 0 6 0 0 6
Fluorescent lac Z 0 6 0 0 6
Sequenced 0 6 0 0 6
Site Direct Mutagenesis (single mutations) 0 4 0 0 4
Yeast Transformation (His, LacZ, FUI) 0 0 0 0
His Growth 0 0 0 0
FUI 0 0 0 0
Fluorescent lac Z 0 0 0 0