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Jason Kahn: G proteins and GPCRs II 1

G-Proteins and GPCRs II Review of G-protein regulation

GPCR dimerization and networking

GPCR Pharmacology

Ras proteins: soluble GTP-binding proteins

Connections among pathways

Note: Vision disobeys all the rules!

Note 2: Need to add 15 minutes of case studies!

Sources: Gomperts, Voet and Voet

Science’s STKE

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Review Regulation of G-proteins When GDP is bound to G-proteins, they are inactive,

when GTP is bound, they are active. Therefore theG-proteins have built-in molecular egg timers. Gα and Gβγsubunits can have independent effects.

In some cases, like EF-Tu, the egg timer enforces fidelity,in our current context it ensures that the signal lasts onlya short time.

GEFs potentiate guanine nucleotide exchange, typicallyGTP binding and hence activation. GPCRs are GEFs.

GAPs and RGS proteins speed up the GTPase activity ofthe G-protein a subunit, hence inactivating it.

GDI’s like the Gβγ subunits slow down GDP release, canactivate or repress.

Multiplicity of effects comes from multiplicity of GPCRs,Gα, Gβ and Gγ.

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GPCR Architecture Can be activated by

light, biogenic amines,peptides, glycosylatedhormones, Ca++, orproteases

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GPCR Pharmacology Structure-activity

relationships (SARs)are obtained bymaking variations oflead compounds thatinhibit the GPCR

This is correlated withmutagenesis of thepresumed bindingpocket residuesderived from homologyto previous receptors

As we have discussed,bacteriorhodopsin andrhodopsin aren’t reallymuch help

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Binding of Agonists/Antagonists

Typically small-molecule ligandscontact several TMhelices

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Receptor Dimerization

GαSSTR5 R

S

Gα

SSTR5 del C

somatostatin

+SSTR5 del C X

Ssomatostatin

+

Ssomatostatin

+

+D2 receptor

Recovery of SS Signaling via D2R

Functional D2R SSTR5 dimer

From STKE

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Receptor Networks Receptor mosacis can

include both homotypicand heterotypicinteractions tointegrate output.

Positive cooperativityallows for a sensitiveresponse to weaksignals

Negative cooperativityallows for protectionagainst elevatedagonist levels and aresponse over a widerange of concentration.

See Agnati et al.,(2005) TiBS 30, 188.

The “signalosome” Does this remind you

of anything?

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Ras Proteins Prototype of soluble G-

proteins

Mutations that lead topersistent GTP binding areoncogenic

Activates MAP kinasepathway, which leads totranscription of genesrequired for proliferation

Homologues in yeast, fly arevery similar

Localized to plasmamembrane by farnesylation

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Ras Recruitment

V+V 3/e

Grb2

SH3-peptide structure

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Ras Mediation of RTK Signalling The Grb2 adaptor protein has SH2 domains that

recruit it to phosphorylated tyrosine residues on RTKs,including the EGF receptor.

The insulin receptor recruits Grb2 indirectly: thephosphorylated InsR recruits Shc through Shc’s SH2domain and then Tyr-phosphorylates Shc to make it aligand for the SH2 domain of Grb2.

Grb2 has two SH3 domains, which bind proline-richbinding motifs on the mSos protein (mammalianhomolog of the Drosophila Son of Sevenless adaptor,where Sevenless is an RTK that regulatesdevelopment of the R7 photoreceptor cell). So Sos isrecruited to the plasma membrane (Sos and Grb2 arestably associated).

mSos is the GEF for Ras, so Ras is recruited to themembrane and activated

In contrast, the platelet-derived growth factor PDGFrecruits RasGAP, which activates the GTPase activityof Ras. Common oncogenic Ras mutants areinsensitive to RasGAP, hence always on.

Ras activates the MAP kinase proliferation pathwaythrough interaction with the effector Raf, a Ser/Thrkinase.

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Guanine Nucleotide Exchange

Structure of Ras/Sos complex

SCR domains partially competewith GDP/GTP binding,reducing G binding affinity andaccelerating kinetics ofexchange.

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The Left Hand of Insulin Signalling

Insulinsignals fedstate,henceenablesgrowth.

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PI3K is an effector for Ras Recall that PI3K (phosphoinositide 3-kinase) is recruited to phosphorylated IRS1 via its

regulatory p85 subunit, which eventually leads to PKB/Akt activation, inactivation ofglycogen synthase kinase 3β, dephosphorylation of glycogen synthase by PP1, andglycogen synthesis.

Recall further that Akt phosphorylates Bad, therefore decreasing its proapoptotic activity. The p110 catalytic subunit of PI3K is activated by binding Ras•GTP So…Ras activation of PI3K leads to phosphorylation of Bad. Why does this make sense? Ras simultaneously signals proliferation through the MAPK

pathway and also inhibits apoptosis through PI3K. Throw both switches.

PI3K p110 isoforms: RBD = Ras binding domain

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Ras Summary Shields et al., Trends Cell Biol. 2000. The RalGDS connection means that Ras can

activate the GEFs that then activate othersoluble G-proteins.

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Network Architecture Junctions split incoming signals to

several output channels (RTKs, Ras)

Nodes integrate several inputs to givea common output (Adenylate cyclase)

Allows for propagation of a signalthroughout a network or dissipation ofthe signal

Buzzphrase: “Emergent properties ofnetworks”

Jordan et al., Cell 2000.

Will require huge amounts of dataand mathematical modeling tounderstand how this works.

DAG

Ca2+

RTK

c-Raf

MAPK-1,2

MEK-1,2

Grb2

SOSRas

Stimulation of Proliferation

PLC-γ

PKC

AA

cPLA2

IP3

PersistentActivation

GAP

Consequence of Networking: A feedback loop that displays bistability

Bhalla and Iyengar(1999)Science 283:381

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Effector Dimerization Nine isoforms talk to different G proteins, Ca++, PKA, PKC Adenylate cyclases are dimers (at least) Dimerization allows for integration of signals from different

heterotrimeric G proteins, through interaction with either α or βγsubunits.

A molecular “coincidence detector?” (How do scintillation counterswork?)

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Receptor Localization Phosphorylated receptors

recycle via endocytosis ofclathrin-coated vesiclesfollowed bydephosphorylation in theendosome and return to theplasma membrane.

Heterotrimeric G proteinswere observed to relocateto the Golgi apparatus(involved in proteintrafficking) after activation,then recycle to the plasmamembrane afterinactivation. Mechanismentirely unclear. (Akgoz etal., JBC 2004)

Proposed to offer amechanism for rapiddesensitization, mediatedby γ subunit.

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Summary

Networks of GTP-binding proteinsignalling are activated by extracellularsignals through GPCRs and RTKs, viaadaptors, GEF activities.

Many and varied downstream effectorsamplify, split, and integrate signals

Signals are attenuated by GAPactivities, network architecture,receptor phosphorylation, and receptorand G-protein trafficking