1. Macrophage Biology Lab, San Francisco
RNAi II (Continued)
Macrophage Biology Lab, San Francisco
Tamara Roach Bob Rebres
Bill Seaman

2. Thanks
Melissa Kachura David Quan Carrie Wong

3. Use of RNAi to target signaling molecules
C5a
UDP
IgG2a
P2Y6
P2Y2
FcgRI
C5aR
Fcg
Gai2 / Gai3
Gaq /Ga11
ITAM
ITAM
PTKs
Gb 1, 2, 4, 5 + Gg
PI 3-kinases
PLCs
Ca2+
PIP3
P - Akt

4. Summary of shRNA-transduced RAW264.7 lines
37 Lines have been studied, covering 31 targets (4 new/week)
Using prescreened shRNA plasmids, target knock-down was
>75% in almost all transduced lines (protein or mRNA)
>90% in two-thirds
>99% in one-third
Apart from receptor knock-downs, alterations in signaling
were observed in:
Half of responses to IgG2a
One-third of responses to C5a
Only one response to UDP by 1.5-fold cutoff, but more by stats
Expected phenotypes were usually detected
Unexpected phenotypes were frequent

5. Gaq facilitates signaling through P2Y receptors
C5a
UDP
IgG2a
P2Y6
P2Y2
FcgRI
C5aR
Fcg
Gai2 / Gai3
Gaq /Ga11
ITAM
ITAM
PTKs
Gb 1, 2, 4, 5 + Gg
PI 3-kinases
PLCs
Ca2+
PIP3

6. shRNA against Gaq reduced the peak [Ca2+]i response to UDP but increased the response to IgG2a
Expression of Gq
Responses to FXM Ligands
(Western blot)
(Peak [Ca2+]i)
IB: Gq 75 50 37
Gq
Vector
Grk2
Mol.Biol.Lab.

7. shRNA against Gaq reduced the peak [Ca2+]i response to UDP

8. Gb2 is utilized by C5aR C5a UDP IgG2a P2Y6 P2Y2 FcgRI C5aR Fcg
Gai2 / Gai3
Gaq /Ga11
ITAM
ITAM
PTKs
Gb 1, 2, 4, 5 + Gg
Gai (and Gao) families use Gb2 to activate PLC
Gaq activates PLC directly
PI 3-kinases
PLCs
Ca2+
PIP3

9. shRNA against G2 inhibited the response to both C5a and IgG2a
G2 expression
Responses to FXM Ligands
(Western blot)
(Peak [Ca2+]i)
IB: G2 49 38 28
Vector
G 2
PTEN
Mol.Biol.Lab

10. The C5a signaling defect was observed in each of 3 G2-shRNA lines

11. The IgG2a signaling defect was observed in 2 of 3 G2-shRNA lines

12. The C5a receptor signals through Gai proteins
UDP
IgG2a
P2Y6
P2Y2
FcgRI
C5aR
Fcg
Gai2 / Gai3
Gaq /Ga11
ITAM
ITAM
PTKs
Gb 1, 2, 4, 5 + Gg
PI 3-kinases
PLCs
Ca2+
PIP3

13. shRNA against Gi2 enhanced the response to C5a
Gi2 Expression
Responses to FXM Ligands
(Western blot)
(Peak [Ca2+]i)
IB: Gi2
Vector
PTEN
Gi2A
Gi3
Mol.Biol.Lab

14. The enhanced [Ca2+]i response to C5a was observed in each of 2 Gi2-targeted lines
Gai2
100nM
C5a
Vector
(HBSS)
100
(nM)
Line #2 ] i 2+ 50
[Ca 50
100
150
Time

15. Pertussis toxin inhibits both Gai2 and Gai3
C5a
UDP
IgG2a
P2Y6
P2Y2
FcgRI
C5aR
Fcg
Gai2 / Gai3
Gaq /Ga11
ITAM
ITAM
PTKs
Pertussis
toxin
Gb 1, 2, 4, 5 + Gg
PLCs
PI 3-kinases
Ca2+
PIP3

16. The [Ca2+]i response in Gi2-targeted cells was partially resistant to pertussis toxin
100 nM C5a

17. High concentrations of pertussis toxin failed to eliminate the [Ca2+]i response to C5a in Gi2-targeted cells
(ng/ml)

18. The next step: targeting both Gai2 and Gai3
C5a
UDP
IgG2a
P2Y6
P2Y2
FcgRI
C5aR
Fcg
Gai2 / Gai3
Gaq /Ga11
ITAM
ITAM
PTKs
Gb 1, 2, 4, 5 + Gg
PI 3-kinases
PLCs
Ca2+
PIP3

19. Overview: RNAi targeting of G proteins and regulators: Both gain of function and loss of function are observed

20. RNAi targeting G proteins and regulators: Both expected and unexpected results were obtained

21. RNAi targeting InsP and phosphoinositide metabolism: Both expected and unexpected results were obtained
= “Expected”
= “Unexpected”

22. RNAi targeting tyrosine kinases and scaffold proteins: Both expected and unexpected results were obtained
= Expected
= Unexpected

23. RNA-mediated RNAi: multiple phenotypes for C5a and IgG2a, but not for UDP

24. C5a, UDP and IgG2a stimulate distinct patterns of intracellular Ca2+ flux
IgG2a+ anti-IgG
Ligand

25. RNA-mediated RNAi: multiple phenotypes for C5a and IgG2a, but not for UDP

26. Summary of shRNA-transduced RAW264.7 lines
37 Lines have been studied, covering 31 targets (4 new/week)
Using prescreened shRNA plasmids, target knock-down was
>75% in almost all transduced lines (protein or mRNA)
>90% in two-thirds
>99% in one-third
Apart from receptor knock-downs, alterations in signaling
were observed in:
Half of responses to IgG2a
One-third of responses to C5a
Only one response to UDP by 1.5-fold cutoff, but more by stats
Expected phenotypes were usually detected
Unexpected phenotypes were frequent

27. Approaches to validation of RNAi phenotypes
Replicate lines with different shRNAs
Alternate KD strategy (antisense, siRNA)
Microarrays to look for off-target effects
Knockdown reversal
Test (and confirm) a likely hypothesis (e.g., with a 2nd knockdown)

28. Conclusions RNAi can be efficiently used in RAW264.7 cells
Results can be replicated
Lines can be expanded
Expected phenotypes are usually detected
Unexpected and interesting phenotypes are frequent

29. Thanks
Rick Brown, UCSF