Departmental Student Presentation Steven A. Moore Jr.

1. Departmental Student Presentation Steven A. Moore Jr. Identification of Amino Acid Motifs in the Human Angiotensin II Type-2 Receptor Involved in G-protein Activation Honors Biology Presentation 2002 Weyhenmeyer Laboratory Dept. of Cell and Structural Biology University of Illinois 512 Medical Science Building (217) 333-8075 samoore@students.uiuc.edu

2. Steve’s Thesis Problem: What are the primary, secondary, and tertiary structures in the human AT2 receptor that are necessary for G-protein coupling and activation?

3. Steve’s Thesis Problem: What are the primary, secondary, and tertiary structures in the human AT2 receptor that are necessary for G-protein coupling and activation? Background Information:

4. Concept Map Angiotension II Peptide Hormone Angiotensin II Type-1 Membrane Receptor Angiotensin II Type-2 Membrane Receptor G-protein Coupled Membrane Receptor Superfamily

5. Introduction to Angiotensin II Hormone • (Ang II) • Angiotensin II octapeptide is a naturally occurring hormone: • Asp-Arg-Val-Tyr-Ile-His-Pro-Phe • Sar1Ile8-Angiotensin II is a synthetic version of Angiotension II : • Sar-Arg-Val-Tyr-Ile-His-Pro-Ile • Best known for blood pressure regulation, body fluid homeostasis, and electrolyte balance (AT1R mediated). • Also found to influence cell growth, differentiation, and death.

6. The G-Protein Coupled Receptor(GPCR) Superfamily • All G-protein receptors are homologous, this has become clear from DNA sequencing experiments. • The amino acid sequences of a large number of these receptors reveal a common structure consisting of a single polypeptide chain that threads back and forth across the lipid bilayer seven times. • ·Common structural motifs found in all GPCRs include: • 1.) Seven a-helical hydrophobic transmembrane regions • 2.) Three extracellular loops and tail • 3.)Three intracellular loops and tail • 4.)A variant of the DRY motif, a highly conserved motif thought to be involved in • G-protein coupling.

7. Fig. 2)Diagram of the human AT2 receptor including the seven transmembrane domains along with intra and extra-cellular loops and tails. Proposed amino acids to be mutagenized are highlighted and their single letter designations are adjacent.

8. Properties of Angiotensin II Receptor Subtypes AT1AT2 Agonist:Ang II Ang II Localization: liver, cortex, HTSM, adrenal gland, uterus, lung, heart, spleen, kidney, heart, pancreas, uterus, adrenal gland, cerebellum, HTSM, v.s.m, kidney cerebral vessels Peripheral function: vasoconstriction, growth, development, adrenal aldosterone wound healing and catecholamine release, enhanced NE release from sym. ganglia

9. AII Step 1)Angiotensin II binding to high affinity site, and subsequent AT2 receptor activation. plasma membrane AII G GDP Step 2)Newly assembled G-protein binds active AT2 receptor. AII Step 4)hormone is released from low affinity binding site after G-protein binds to active receptor. AII G Step 3)GDP exchanged for GTP GDP GTP GDP Inactive receptor Step 5) Active G-protein activates next enzyme in pathway. G ? ? GDP Fig 1.) G-protein activity diagram.

10. Introduction to Angiotensin II type-2 Receptor (AT2R) ·Member of the G-protein Coupled Receptor(GPCR) superfamily. ·Agonist: Angiotensin II Hormone Partial Agonist: Sar1Ile8-Angiotensin II Specific Antagonist: PD123,319 ( blocks the receptor for Angiotension II) ·Major structural motifs include: 1.) 7 hydrophobic transmembrane regions. 2.) Hormone binding regions comprised of 3 extracellular loops and tail. 3.) G-protein binding regions comprised of 3 intracellular loops, including the DRY motif, and cytoplasmic tail.

11. Fig. 2)Diagram of the human AT2 receptor including the seven transmembrane domains along with intra and extra-cellular loops and tails. Proposed amino acids to be mutagenized are highlighted and their single letter designations are adjacent.

12. Introduction to Angiotensin II type-2 Receptor (AT2R) ·Member of the G-protein Coupled Receptor(GPCR) superfamily. ·Agonist: Angiotensin II Hormone Partial Agonist: Sar1Ile8-Angiotensin II Specific Antagonist: PD123,319 ( blocks the receptor for Angiotension II) ·Major structural motifs include: 1.) 7 hydrophobic transmembrane regions. 2.) Hormone binding regions comprised of 3 extracellular loops and tail. 3.) G-protein binding regions comprised of 3 intracellular loops, including the DRY motif, and cytoplasmic tail. ·AT2 receptor has been shown to be involved in: Inhibition of cell proliferation (Tsuzuki et al., 1996) Development (Mukoyama et al., 1993) Apoptosis (Yamada et al., 1996) Neuronal differentiation (Schelman et al., 1997, Bedecs et al., 1997)

14. Fluorescent Stained Neuron

15. Steve’s Thesis Problem: What are the primary, secondary, and tertiary structures in the human AT2 receptor that are necessary for G-protein coupling and activation? Hypothesis: 1.) If the DRY motif (Asparagine, Arginine, Tyrosine), found in the human AT2 receptor, is a key player in G-protein activation, then mutation of this motif should significantly effect the receptors ability to activate G-proteins. 2.) If a specific sequence of the AT2 receptor is key to binding G-proteins, then we can identify regions by using synthetic proteins to compete with the active receptor for the G-protein.

16. Experiments to test Hypotheses: • ·1a). Determine whether mutations in the DRY motif of the AT2 receptor effect AngII hormone binding affinity or receptor expression levels? • 1b.) Determine whether mutations in the DRY motif effects the receptor’s • ability to bind and activate G-proteins. • ·2.)  Map the G-protein binding domains of the AT2 receptor using synthetic peptides selected from the receptor sequence.

17. 1a.) Mutation, transfection, and characterization of wild type and DRY motif mutant receptors. Site-directed mutagenesis of human AT2 receptor. (PCR based method) Insert wild type and mutant receptor DNA into Chinese Hamster Ovary cells. (Lipid based transfection protocol) Radioactively tagged ligand binding analysis of wild type and mutant receptors. Scatchard analysis will be used to calculate binding affinity and expression levels for each receptor.

21. CHO Wt D141-A R142-A Y143-A DRY-AAA Western blot of CHO cells transfected with wild type and mutant AT2 receptors: Lane 1.): non-transfected CHO-K1 cells, 2.) Wild type human AT2 receptor, 3.) D141-A mutant receptor, 4.) R142-A mutant receptor, 5.) Y143-A mutant receptor, 6.) DRY-AAA mutant receptor

22. 1b.) Effects of GTPgS on AT2 receptor's Ang II binding affinity. • ·GTPgS will decrease a G-protein coupled receptor's affinity for its ligand by maintaining the receptor in an active state. • ·Wild type receptor is expected to have the largest decrease in Ang II affinity with the addition of GTPgS. Wild type receptor % decrease of Ang II affinity • with GTPgS will be the positive control.

23. AII Step 1)Angiotensin II binding to high affinity site, and subsequent AT2 receptor activation. plasma membrane AII G GDP Step 2)Newly assembled G-protein binds active AT2 receptor. AII Step 4)hormone is released from low affinity binding site after G-protein binds to active receptor. AII G Step 3)GDP exchanged for GTP GDP GTP GDP Inactive receptor Step 5) Active G-protein activates next enzyme in pathway. G ? ? GDP Fig 1.) G-protein activity diagram.

25. 2.) Use of synthetic peptides to identify key regions of • G-protein coupling. • ·If a specific sequence of the AT2 receptor is key to binding G-proteins, then we • can identify regions by using synthetic proteins to compete with the active receptor for the G-protein. • ·This protocol has also been used to map G-protein coupling regions in the b-adrenergic (Strader et al., 1987; O'Dowd et al., 1988; Munch et al., 1991), a1-adrenergic (Cotecchia et al., 1990), rhodopsin (Konig et al., 1989), and the m2 muscarinic receptors (McClue et al., 1994). • ·Advantages and Disadvantages vs. Site-directed Mutagenesis

27. Conclusions • 1a.) • ·Wild type receptors can be mutated via a PCR based site-directed mutagenesis protocol. We have also demonstrated that high transient expression levels can be obtained using a lipid based transfection protocol. • ·Even more importantly, we have been able to determine that the mutations in the DRY motif did have a significant effect on the receptor's Ang II affinity, and in some cases expression levels. • 1b.) • ·We have shown that GTPgS decreases Ang II affinity in all of the receptors tested. This demonstrates that all receptors are coupling with G-proteins. As expected, the wild type receptor had the largest decrease of affinity, signifying it's strong affinity for G-proteins. • 2.) • We have demonstrated that we can map key G-protein coupling regions • using synthetic proteins to compete with active receptor.

28. Thanks: James Weyhenmeyer, Ph.D. Anjali Patel Nancy Huang Bridget Lavin Jungsik Yo Paul Ferguson Tom Grammatopoulos Rob Andres

29. Steve’s Lab

30. Steve’s Poster Presentation

31. Incubating the CHO

32. Examining Cells

33. Feeding the CHO

34. Frozen CHO

35. It’s a Radioactive Lab!

36. More expensive equipment

37. Steve’s Lab Slave! (He wishes!)

38. Biohazards!

39. Rule # 1 of good lab technique. Cleanliness!

40. Macs Rule! PC’s Drool

41. Where Grad students hang out

42. A PhD in Molecular Biology does not qualify you for a low level maintenance position.

43. High level discussions