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

Thanks Melissa Kachura David Quan Carrie Wong

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

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

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

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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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)

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

Thanks Rick Brown, UCSF