1. A regulator of G protein signaling interaction surface linked to effector specificity 4/24/2008

2. Heterotrimeric G Proteins Secondary messenger protein in various tissues from the heart to the eye Important for drug development “GPCRs (G-Protein Coupled Receptors) are targeted by 12 of the top 20 selling drugs, including Coreg for congestive heart failure, Cozaar for high blood pressure, Zoladex for breast cancer, Buspar for anxiety and Clozaril for schizophrenia, as well as by Zantac and Claritin. Together the drug class accounts for $200bn (€159 bn) in annual sales.” - Chu, 2006

3. G Proteins continued Regulator of G Protein Signaling (RGS) increases rate of GTP hydrolysis by G  -GTP, speeding up system

4. How is RGS specificity maintained? There are over 50 RGS proteins identified with a variety of activities and sequences. Crystal structure shows contact residues are highly conserved between RGS and G  suggesting other partners. What region provides this specificity?

5. Evolutionary Trace Method Input: Aligned protein family sequences, evolutionary tree and protein structure Output: Ranking of evolutionary importance visually represented on the protein structure

6. Evolutionary Trace Method 1) Subdivide protein family sequence into groups based on evolutionary tree branches

7. E.T. Method Continued 2) At each trace level, refer back to the alignment and report the conservation of residues within each subgroup

8. E.T. Method Continued 3) If a position is invariant in a subgroup but differs between subgroups. It is a Class-Specific residue.

9. E.T. Method Continued This process can be repeated until all positions receive a rank. Residues with lower ranks are considered more important.

10. ET of RGS Protein Family Result of ET method yields initial contact residues and putative binding surface

11. Evolutionarily Privileged Surface of the RGS Domain Secondary Structure Residues are non- contiguous but cluster spatially Surface containing both invariant and class-specific residues

12. Cluster of class-specific residues at the RGS-G  interface RGS protein in white G  shown in yellow Invariant in red Class specific blue B is the newly identified residues, shape suggests location for binding

13. Correlation of ET identified RGS residues with PDE-y effects RGS proteinPDE-y effect* ET identified residue number (based on the RGS4) 77117121122124126134 RGS4InhibitLysGlu-ValGlnThrGluArg RGS16InhibitHisGlu-SerGluProGluArg GAIPInhibitArgAsp-IleLeuProGluArg RGS6InhibitLeuGlu-ProGlyProAlaTyr RGS9EnhanceGlnLeuProGlyArgTrpMet RGS11NoneMetGlnProGlyAlaTrpMet Example: 117, - inhibits, non-polar enhances, non-charged polar has no effect

14. Modulation of RGS GAP by PDEy GTPase accelerating protein (GAP) modulation is a phenotype used to compare taxa

15. Class specific residues cluster near putative PDE-y binding site When mapped to the structure, the class- specific residues are located on a contiguous range providing a putative PDE-y binding site

16. Conclusions The evolutionary trace method can identify functionally important residues within a protein structure. The ET of the RGS protein family confirmed 10 of 11 previously identified contact residues and discovered a cluster of class specific residues which could function as a binding domain for PDE-y to control RGS GAP activity.