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