2. Project 2: Cellular Mechanisms and Interaction of Neurokinin-1 Receptor (NK- 1R) Antagonists and HIV Co- Receptors CCR5 and CXCR4 Principal Investigator - S.D. Douglas, M.D. Co-Investigators -L.E. Kilpatrick, Ph.D J.-P. Lai, M.D.

3. SP and NK-1R in Macrophages SP is a mediator of neuroimmuno- regulation Macrophages synthesize SP and express SP specific receptors (NK-1R)- autocrine loop SP enhances HIV infection of macrophages NK-1R antagonists inhibit HIV infection of macrophages

4. The chemokine receptors CCR5 and CXCR4 are the primary co-receptors for HIV infection NK-1R antagonists selectively inhibit HIV strains which use CCR5 as a co- receptor Thus, there is a critical relationship between NK-1R and CCR5 receptors Role of CCR5 in HIV Infection of Macrophages

5. CCR5 NK-1R Signal Transduction? Hetero- dimerization? Protein Synthesis? CXCR4 HIV Cytokines, Chemokines, CCR5, CXCR4 NK-1R Antagonists NK-1R:CCR5 Interaction in Macrophages CD4 SP

6. Goal of Project 2 To determine the mechanism by which SP:NK-1R regulates CCR5 function 1)GPCR crosstalk at the level of the receptor or between signaling pathways 2)Direct effect on CCR5 expression through alterations in receptor synthesis 3)Indirect interactions through cytokines and chemokines

7. The Specific Aims of this Project are designed to examine: Selectivity of NK-1R Antagonists for CCR5 Mechanism of Interaction between CCR5 and NK-1R Level of Receptor (heterodimer complexes) Signaling Pathways (crosstalk) Regulation of CCR5 expression Regulation of cytokines and chemokines Specificity of NK-1R Antagonists (siRNA)

8. Specific Aim 1 We will characterize the antiviral and immunomodulating effects of Aprepitant and other candidate NK-1R antagonists on CCR5 function. We will ascertain whether the interaction between NK-1R and CCR5 is unique to the CCR5 receptor or a common feature of chemokine receptors, chemotactic receptors, or GPCRs.

9. In vitro Inhibition of Pseudotyped HIV infection of Macrophages by NK-1R Antagonists

10. Aim 1: Characterization of the effect of NK-1R Antagonists on CCR5 receptor-mediated functions NK-1R antagonists Macrophages Untreated controls 1.Antiviral: Infection by pseudotype HIV (ADA) 2.Immunomodulatory: Chemotaxis to ligands of CCR5, CXCR4, FPR, CSF-1 3.Immunomodulatory and Antiviral: Expression (protein and mRNA) of CCR5, CXCR4, chemokines, and cytokines

11. Test the hypothesis that NK-1R antagonists alter CCR5 mediated physiological responses through G-protein coupled receptor (GPCR) crosstalk. Specific Aim 2

12. Enhancement of RANTES mediated Ca 2+ Mobilization by SP

13. Aim 2: NK-1R antagonists alter CCR5-mediated physiological responses through receptor crosstalk SP and/or NK-1R antagonists Untreated controls 1. Heterodimers of NK-1R and CCR5 (Constitutive and agonist-mediated) 2. Receptor phosphorylation and G-protein coupling 3.Receptor internalization and desensitization Macrophages

14. Specific Aim 3 Test the hypothesis that activation of NK-1R mediated signaling amplifies CCR5 mediated intracellular signaling resulting in synergistic crosstalk between NK- 1R and CCR5 signal transduction pathways.

15. Buf SP R SP+R SP+R+A Phospho-p44-MAPK- Phospho-p42-MAPK- p44-MAPK- p42-MAPK- Synergistic Activation of p44/42 MAPK by RANTES and SP

16. Aim 3: NK-1R antagonists interrupt positive crosstalk Between NK-1R and CCR5 receptors SP and/or NK-1R antagonists Untreated controls 1. CCR5-mediated Ca 2+ mobilization and PKC activation 2. CCR5-mediated activation of MAP kinases and NF  B 3. Specificity of NK-1R for CCR5 vs. CXCR4, FPR, CSF-1 Macrophages

17. Specific Aim 4 We will further define the specificity of Aprepitant and other NK-1R antagonists for the NK-1R receptor through knockout studies using NK-1R siRNA in the macrophage cell line THP-1.

19. Aim 4: The relationship between NK-1R and CCR5 (as determined by NK-1R Knockout) Untreated controls (NK-1R, CCR5, CXCR4) NK-1R Knockout cells (CCR5 and CXCR4) 1. Infectivity of THP-1 cells with R5 or X4 HIV strains 2. CCR5/CXCR4 expression 3. CCR5/CXCR4 signaling and chemotaxis

20. Interaction with other Projects and Cores Project 2 (Douglas, Kilpatrick) Core B (BBI)Core C (Biostatistics) Project 3 (Lackner) Project 1 (Ho) Project 4 (Tebas) Data analysis and PKA Effects of Chronic Antagonist Exposure Immuno- modulatory effect Immuno- modulatory effect NK-1R-CCR5 interaction