2. 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

3. 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 Assay6) PCR 2) His Growth Assay7) Western Blot 3) Plasmid Manipulation8) Sequencing 4) Restriction Digest9) Mutagenesis 5) Fluorescent Lac Z10) 3-D Modeling

4. 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

5. 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.

6. 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

7. 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

8. 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.

9. Fluorescent Lac Z Same principle as filter lift. FDG is used as substrate in place of X Gal. When cleaved, FDG fluoresces.

10. 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.

11. Sequencing DNA is sent to be sequenced. Results where mutations, if any, are present. Mutations that we found:Mutation Rate: Revert 2-13: V82G1/1089 Revert 2-16: V82G1/1089 Revert 2-21: M63L, S312P2/1089 Revert 2-26: S123I, E288G4/1089 Revert 2-32: F29S, V59A, F248I, V320E4/1089 Revert 2-49: F248I 2/1089 Overall Mutation Rate: 14/6532=.21%

13. Mutagenesis Site directed mutagenesis of CXCR4 CAM Separates multiple mutations Mutate into pcDNA3.1zeo(+) for transfection into Mammalian Cells Mutations: M63L V82G F248I E288G

14. 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

16. PLATE 1 PLATE 2 PLATE 3 PLATE 4 PLATE 5 PLATE 6 PLATE 7 PLATE 8 PLATE 9TOTAL Filter Lift51444745 48494748424 His Growth036473844410400246 Yeast Miniprep033432143310370208 Bact Transformation0650011 Bact MinipreP0650011 Sal 1 and bact transf. Miniprep0650011 Yeast TRAnsformation (lac Z)06006 Fluorescent lac Z06006 Sequenced06006 Site Direct Mutagenesis (single mutations)04004 Yeast Transformation (His, LacZ, FUI)0000 His Growth0000 FUI0000 Fluorescent lac Z0 0 00