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.

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

• 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

CCR5NK-1R

Signal Transduction?

Hetero-dimerization?

Protein Synthesis?

CXCR4

HIV

Cytokines, Chemokines, CCR5, CXCR4

NK-1RAntagonists

NK-1R:CCR5 Interaction in Macrophages

CD4

SP

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

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)

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.

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

Antagonists

0

20

40

60

80

100

120

RL

U (

% o

f C

on

tro

l)

Control Aprepitant CJ12,255 CP-96,345 RP67,580 L-733060

10-6M 10-6M 10-6M 10-6M 10-6M

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

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

Specific Aim 2

Enhancement of RANTES mediated Ca2+ Mobilization by SP

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

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.

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

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 Ca2+ mobilization and PKC activation

2. CCR5-mediated activation of MAP kinases and NFB

3. Specificity of NK-1R for CCR5 vs. CXCR4, FPR, CSF-1

Macrophages

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.

Knockdown NK-1R in NK-1R transfected THP-1 Cells using siRNA (48 hours post transfection)

0

20

40

60

80

100

120

NK-1R siRNA-147 siRNA-742 siRNA-442 siRNA-547

Rel

ativ

e N

K-1

R m

RN

A l

evel

s (%

)

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

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 PKAEffects of Chronic Antagonist Exposure

Imm

uno-

mod

ula

tory

eff

ect

Imm

uno-

modulatory effectNK

-1R-C

CR5 inte

ract

ion