1. Grave’s Disease
CTLA4
Keith Christensen

2. What is Grave’s disease?
Thyroid
eyes

4. Genetic structure of CTLA4
Leader peptide
Immunoglobulin
V-Domain
Trans-
mem
Cytoplasmic tail
49 A/G mutation

5. CTLA4 has strong homology among mammals
Human
Chimpanzee
Dog
Cow
Chicken
Trout
*Mouse
CTLA4
Immunoglobulin V-domain Tm
Ctyo
CTLA4
Immunoglobulin V-domain Tm
Ctyo
98%
88%
84%
48%
34%
75%
CTLA4
Immunoglobulin V-domain Tm
Ctyo
CTLA4
Immunoglobulin V-domain Tm
Ctyo
CTLA4
Immunoglobulin V-domain Tm
Ctyo
CTLA4
Immunoglobulin V-domain Tm
Ctyo
CTLA4
Immunoglobulin V-domain Tm
Ctyo

6. Phylogeny of CTLA4

7. Gene ontology of CTLA4
Molecular: protein binding Biological: immune response Cellular: membrane associated
Include mouse comparrison?
Include a network map

8. What is the function of CTLA4?

9. The trafficking of CTLA4 is highly dynamic

10. How does the 49A/G mutation alter CTLA4?
Leader peptide
Immunoglobulin
V-Domain
Trans-
mem
Cytoplasmic tail
Threonine → Alanine

11. Hypothesis: The 49A/G mutation impairs surface expression of CTLA4 in T lymphocytes by interrupting cellular trafficking
WT A/G

12. What proteins are involved in the trafficking of CTLA4?
eyes

13. Homologs have similar protein interactions
Human
Mouse

14. Possible proteins affected by 49A/G mutation

15. Does the 49 A/G mutation alter the interaction with trafficking proteins?
Eyes

16. Interaction with known trafficking proteins
Reporter gene
DBD
CTLA4
AP2 AD
Reporter gene
With identified
DBD
49A/G
AP2 AD

17. Interaction with novel trafficking proteins
Reporter gene
DBD
CTLA4
Prey AD
Reporter gene
novel
DBD
49A/G
Prey AD

18. Are the trafficking proteins regulated by the immune response?
eyes

19. Altered expression during immune stimulation
Wild type
49 A/G
Resting Activated
Resting Activated
CTLA4
CTLA4
CD28
CD28
AP2
AP2
Modifier
Modifier
Expression Level: High Low

20. Are post translational modifications responsible for the disruption in protein binding?
eyes

21. Use MALDI-TOF to identify post translational modifications
Leader peptide
Immunoglobulin
V-Domain
Trans-
mem
Cytoplasmic tail
Glycosylation Phosphorylation
Stimulated versus Resting

22. Can the phenotype of defective CTLA4 trafficking be remediated?
eyes

23. Use a chemical genetic screen
Screen: monitoring surface CTLA4 Phenotype: Mouse survival

24. Future Directions
Link protein modification to protein interaction Endosome/lysosome Apply to autoimmune model
eyes

25. Anjos S, Nguyen A, Ounissi-Benkalha H,Tessier MC, Polychronakos C A common autoimmunity predisposing signal peptide variant of the cytotoxicT-lymphocyte antigen 4 results in inefficient glycosylation of the susceptibility allele. J. Biol. Chem. 277:46478–86
April K.S. Salama and F. Stephen Hodi Cytotoxic T-Lymphocyte Antigen-4. Clin Cancer Res; 17:
Omar S. Qureshi‡1, Satdip Kaur‡2, Tie Zheng Hou‡3, Louisa E. Jeffery‡4, Natalie S. Poulter§, Zoe Briggs, et al Constitutive Clathrin-mediated Endocytosis of CTLA-4 Persists during T Cell Activation. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 12, pp. 9429–9440, DOI /jbc.M
Teft, W. A., Kirchhof, M. G., and Madrenas, J. (2006) A molecular perspective of CTLA-4 function. Annu. Rev. Immunol. 24, 65–97
Chuang, E., Alegre, M. L., Duckett, C. S., Noel, P. J., Vander Heiden, M. G., and Thompson, C. B. (1997) Interaction of CTLA-4 with the clathrinassociated protein AP50 results in ligand-independent endocytosis that limits cell surface expression. J. Immunol. 159, 144–151
Shiratori, T., Miyatake, S., Ohno, H., Nakaseko, C., Isono, K., Bonifacino, J. S., and Saito, T. (1997) Tyrosine phosphorylation controls internalization of CTLA-4 by regulating its interaction with clathrin-associated adaptor complex AP-2. Immunity 6, 583–589
Lindsten, T., K. P. Lee, E. S. Harris, B. Petryniak, N. Craighead, P. J. Reynolds, D. B. Lombard, G. J. Freeman, L. M. Nadler, G. S. Gray, et al Characterization
of CTLA-4 structure and expression on human T cells. J. Immunol. 151:3489.
Chistiakov D., Turakulov R., (2003). CTLA-4 and its role in autoimmune thyroid disease. J Mol Endocrinol, 31, doi: /jme
National Endocrine and Metabolic Diseases Information Service. (2012). Grave’s Disease. Retrieved February 7, 2013 from 

26. Questions?