Protein-Protein Interaction Motifs in GPCR Presented by: Harpreet K Dhiman Research Associate, Dept. of Pharmacology, Un

1. Protein-Protein Interaction Motifs in GPCRPresented by:Harpreet K DhimanResearch Associate,Dept. of Pharmacology, Univ. of Pittsburgh May 13, 2003

2. Motifs in GPCR Introduction to Protein-Protein Interactions in GPCR May 13, 2003

3. Motifs in GPCR GPCR Interactions Occur in Cytoplasmic Loop 3rd 7th 11th 15th May 13, 2003

4. Motifs in GPCR Receptors Divided into Various Classes based on Sequence Homology • GPCR classes: • Class A Rhodopsin like • Class B Secretin like • Class C Metabotropic glutamate / pheromone • Class D Fungal pheromone • Class E cAMP receptors (Dictyostelium) • Frizzled/Smoothened family • Putative Families: • Ocular albinism proteins • Drosophila odorant receptors • Plant Mlo receptors • Nematode chemoreceptors • Vomeronasal receptors (V1R & V3R) • Orphans: • Putative / unclassified GPCRs May 13, 2003

5. Motifs in GPCR Goal • Identify Motifs to Find Potential Protein-Protein Interactions in GPCR’ s May 13, 2003

6. Motifs in GPCR Approach Step 1: List proteins that interact with GPCRs based on experimental “proteomic” approach (affinity chromatography and mass spectrometry) Step 2: Identify domains in these proteins using Swiss-prot (web based tool) Step 3: Select domains that have known protein-protein interaction modules Step 4: Identify general Motifs for PDZ and SH3 domains from research articles May 13, 2003

7. Motifs in GPCR Step 1: List Proteins that Interact with GPCRs based on experimental “proteomic” approach (affinity chromatography and mass spectrometry) • VeLi3 • Post-synaptic density protein 95 (PSD95) • Dlgh3 protein (MPP3) • Calmodulin • F-actin capping protein β subunit (CAPZ β) • F-actin capping protein α-2 subunit (CAPZ α-2) • PKC θ-interacting protein PICOT • Actin, cytoplasmic 1 (β-actin) • 2810409H07Rik protein • Dynamin 1 • SPectrin α II chain (α-fodrin) Source: Synaptic multiprotein complexes associated with 5-HT2C receptors: a proteomic approach. Becamel et al, The EMBO Journal, Vol 21, No. 10 pp 2332-2342, 2002 May 13, 2003

8. Motifs in GPCR Step 2: Identify Domains in these proteins using Swiss-prot (web-based tool) May 13, 2003

9. Motifs in GPCR Step 3: Select Domains with known Protein-Protein Interaction Modules Green = Aid in protein-protein interaction Red = Don’t aid in protein-protein interaction May 13, 2003

10. Motifs in GPCR SH3 Domain Structure and Binding The basic fold of SH3 domains contains five anti-parallel β-strands packed to form two perpendicular β-sheets. Src-homology 3 (SH3) domains generally bind to Pro-rich peptides that form a left-handed polyPro type II helix, with the minimal consensus Pro-X-X-Pro. Each of these aliphatic-Pro pairs binds to a hydrophobic pocket on the SH3 domain. From this, two classes of SH3 domains have been defined (Class I and Class 2) which recognize RKXXPXXP and PXXPXR motifs respecitvely. The ligand can, in principle, bind in either orientation. Directionality is conferred by the interaction of the Arg or Lys residue with the charged outer face of the SH3 domain while the tandem prolines bind within two hydrophobic pockets of the SH3 domain. The figure shows a Sem5 C-terminal SH3 domain complexed to the mSos-derived sequence PPPVPPRRR. May 13, 2003

11. Motifs in GPCR PDZ Domain Structure and Binding PDZ domains contain ~80-90 residues that fold into a structure with a β-sandwich of 5-6 β-strands and two α-helices. The peptide ligand binds in a hydrophobic cleft composed of a β-strand (βB), an α-helix and a loop that binds the peptide carboxylate group. The peptide binds in an anti-parallel fashion to the βB strand, with the C-terminal residue occupying a hydrophobic pocket. PDZ heterodimers form a linear head-to-tail arrangement that involves recognition of an internal on one of the partner proteins. The figure shows the 3rd PDZ domain of PSD-95 bound to a TKNYKQTSV peptide. May 13, 2003

12. Motifs in GPCR Binding to Internal Motifs: Internal motifs can be recognized if they are presented within a specific tertiary structure context that conformationally mimics a chain terminus. Fig. The same PDZ domain can recognize two structurally distinct ligands. The a1-syntrophin PDZ domain is shown as a gray solvent-accessible surface. (A) The ligand is canonical peptide NH2-VKESLV-COOH, shown as a red tube (Schultz et al., 1998). (B) The ligand is the nNOS PDZ domain with its distinctive b-finger motif (indicated), shown as a red ribbon diagram. The internal peptide motif that mimics a C-terminal peptide is indicated (Hillier et al., 1999). Source: http://www.ucsf.edu/limlab/papers/bzh_2001b.pdf May 13, 2003

13. Motifs in GPCR • Internal Motif Recognition • e.g. nNOS-syntrophin complex • Interaction mediated by contacts between PDZ domain of syntrophin and a β-finger in the PDZ flanking region of nNOS • First strand of nNOS β-finger (sequence - ETTF- ) mimics a canonical C-terminal peptide in its sequence specific interaction with the peptide-binding grove of syntrophin’s PDZ • C-terminal free carboxylate group is replaced by sharp β-turn at the tip of β-finger May 13, 2003

14. Motifs in GPCR Internal Motif Recognition (β-finger required) Structure of PDZ and PDZ-like domains A : Ribbon diagram of PDZ3 of PSD-95 complexed with a C-terminal peptide from CRIPT. The structure demonstrates the six β-strands (turquoise) and two α-helices (red) with the peptide (yellow) binding as a β-strand between the α B helix and βB strand. B : Structure of α1-syntrophin PDZ domain complexed to nNOS (green). Note the overall similarity in PDZ structure with replacement of the C-terminal peptide ligand by a β-finger. May 13, 2003

15. Motifs in GPCR Step 4 : Identify general Motifs for PDZ and SH3 domains from Research Articles General Motifs Identified for PDZ Domain May 13, 2003

16. Motifs in GPCR Step 4 contd. General Motifs Identified for SH3 Domain May 13, 2003

17. Motifs in GPCR RESULTS • PDZ domain recognizing motifs were found to be present in all classes of GPCR’s (Class through Class F) • SH3 domain recognizing motifs were found in all classes of GPCR’s except Class E. May 13, 2003

18. Motifs in GPCR May 13, 2003

19. Motifs in GPCR May 13, 2003

20. Motifs in GPCR Some Examples.. Receptors with PDZ domains in Class A May 13, 2003

21. Motifs in GPCR Some Examples.. Receptors with SH3 domains in Class A May 13, 2003

22. Motifs in GPCR Some Examples.. Receptors with PDZ domains in Class B May 13, 2003

23. Motifs in GPCR Some Examples.. Receptors with SH3 domains in Class B May 13, 2003

24. Motifs in GPCR Some Examples.. Receptors with PDZ domains in Class C May 13, 2003

25. Motifs in GPCR Some Examples.. Receptors with SH3 domains in Class C May 13, 2003

26. Motifs in GPCR Some Examples.. Receptors with PDZ domains in Class D May 13, 2003

27. Motifs in GPCR Some Examples.. Receptors with SH3 domains in Class D May 13, 2003

28. Motifs in GPCR Some Examples.. Receptors with PDZ domains in Class E May 13, 2003

29. Motifs in GPCR Some Examples.. Receptors with PDZ domains in Class F May 13, 2003

30. Motifs in GPCR Some Examples.. Receptors with SH3 domains in Class F May 13, 2003

31. Motifs in GPCR Questions ? May 13, 2003

32. Motifs in GPCR Acknowledgements • Judith Klein Seetharaman • Betty Cheng May 13, 2003