Download Small GTPases

Monomeric G proteins
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G proteins = Guanine Nucleotide-Binding Proteins
 Bind GTP in the active conformation and GDP in the inactive conformation
 Catalyze the hydrolysis GTP → GDP + P
Heterotrimeric G proteins
Monomeric G proteins
• 3 subunits (α,,γ)
• = small GTPases: Mr = 20-25 kDa
• activated by G protein-coupled receptors
• not directly activated by GPCRs
• GDP is exchanged for GTP in the
α-subunit → α dissociates from γ and
interacts with the effector; GTP is
hydrolyzed → inactivation
• similarly to the α-subunit, GDP
exchange for GTP leads to interaction
with an effector; GTP is hydrolyzed →
inactivation
Small GTPases
 GDP/GTP-regulated molecular „switches“ in signalling pathways
 Two-state structural change is fundamental for their biological function:
hydrolysis of GTP
GTP-protein
active conformation
higher affinity for the effector
GDP-protein
inactive conformation
low affinity for the effector
GDP exchange for GTP
 Effectors:
• protein kinases
• phosphatidylinositol 3-kinase: converts PIP2 to PIP3 which regulates the
activity of other target molecules
• phospholipases…
Regulatory proteins
 Nucleotide exchange and GTP hydrolysis occur at (slow) basal intrinsic rates
 There are regulatory proteins that change the rate of these processes:
• GEFs (guanine nucleotide exchange factors) increase the rate of nucleotide exchange
• GAPs (GTPase-activating proteins) increase the rate of the GTP hydrolysis
• GDIs (guanine nucleotide dissociation inhibitors) block the activation
Activation:
a signal leading to activation of
GEF → GDP exchange for GTP in
the small GTPase molecule →
interaction with the effector
Classification of small GTPases
 Over 150 human monomeric G proteins have been identified
 This Ras superfamily is further divided into several families on the basis of
sequence and functional similarities; main families: Ras, Rab, Rho, Arf, Ran
Post-translational modification of small GTPases
by prenylation
Prenylation probably facilitates
association of small GTPases with
specific membrane compartments
1. Ras: farnesyltransferase – covalent addition of farnesyl isoprenoid (C15)
to the Cys residue of the C-terminal CAAX motif (X = S, M)
2. Rho, Ras: geranylgeranyltransferase I – covalent addition of geranylgeranyl
isoprenoid (C20) to the Cys residue of the C-terminal CAAX motif (X = L, F)
3. Rab: geranylgeranyltransferase II – covalent addition of geranylgeranyl isoprenoid to the
Cys residues of the C-terminal motifs CC, CXC, CCX, or CCXXX
Guanine Nucleotide Exchange Factors (GEFs)
 Facilitate the exchange of bound GDP for free cytosolic GTP, thus regulating
small GTPase activation
 Distinct families of GEFs act upon distinct families of small GTPases
Diverse signals promote Ras activation by stimulating GEFs
 GEFs often contain a large number of different signalling domains which allow them to
respond to a wide variety of signals:
• Ca2+
• signals acting via binding to receptor tyrosin kinases
• DAG…
 All these signals lead to activation of GEFs through their recruitment to the membrane:
tyrosin kinase receptor
(after dimerization)
signal
(hormone)
activation
Sos = Ras GEF
adaptor protein
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GTPase-Activating Proteins (GAPs)
 Accelerate GTP hydrolysis and thus make the signalling more transient
 Structurally distinct GAPs for distinct families of small GTPases exist
 Mutated Ras proteins found in human cancers (single AA substitution) are
insensitive to GAP stimulation, and therefore persistently GTP-bound (active)
 deregulated effector activation
Guanine Nucleotide Dissociation Inhibitors (GDIs)



Inhibit activation by two negative regulatory functions:
•
1. they bind to and mask the isoprenoid modification in the small GTPase,
thus preventing association of the small GTPase with membranes
•
2. their binding perturbs GEF regulation, preventing small GTPase
activation
Less specific then GEFs and GAPs
Up to now identified only for Rho and Rab
Small GTPase structure
 All small GTPases share the G domain which contains: GTP-binding and hydrolyzing elements, effector (and also GEFs and GAPs) binding elements that change
conformation in the GDP- and GTP-bound states (switch I, II)
 G domain is similar in all small GTPases:
 Divergent sequence composition of switches I, II among small GTPases contributes
to the specificity of different small GTPases for the different binding partners
Diverse functions of small GTPases
 Small GTPases facilitate a remarkably divergent spectrum of cellular functions,
which is accomplished by:
• the input signals that regulate the distinct GAPs and GEFs
• unique set of effectors that are recognized by small GTPases
 Overview of the principal functions of small GTPases:
• Ras: regulation of cell proliferation and differentiation
• Rab, Arf: modulation of membrane trafficking
• Ran: regulation of the nucleocytoplasmic transport
• Rho: regulation of actin cytoskeletal organization
Ras family
 Function: regulation of cell proliferation and differentiation
 In various human tumours, mutations in Ras proteins were found that block
intrinsic and GAP-stimulated GTP hydrolysis and result in Ras protein being
locked in the active GTP-bound state
 Signal pathway regulating cell proliferation:
Grb2,SHC – adaptor proteins (recruit Sos)
Sos = Ras GEF
Raf, MEK, MAP – Ser/Thr protein kinases
TF = transcription factor; after phosphorylation, it activates gene expression
Ras proteins as signalling nodes
 1. Here, a wide diversity of extracellular signals converge on (and activate Ras):
• growth factors (epidermal growth factor)
• hormones (insulin)
• cytokines
• extracellular matrix proteins (via integrins)
 2. Activated Ras regulates the activities of effectors with highly divergent functions:
• Raf (Ser/Thr kinase)
• phosphatidylinositol 3-kinases
• GEFs of other small GTPases
• PLCε – cleaves PIP2 to IP3 and DAG (second messengers)
Rab family
 Function: membrane trafficking: Rabs are implicated in vesicle budding from
the donor membrane, transport along the cytoskeleton, docking to, and fusion
with the acceptor membrane
 The largest family of small GTPases (at least 60 proteins in human)
 Localization: organelles of the secretory and endocytic pathway:
• Golgi
• endosomes
• melanosomes
• secretory granules of mast cells…
Cellular localization of Rabs
1. Endocytosis: internalization in clathrin-coated
vesicles (CCV) → delivery to the early/sorting
endosomes (SE); cargo is either sorted for transport
along the degradative pathway to late endosomes
(LE) and lysosomes (Lys), or recycled to the plasma
membrane (directly from SE or indirectly via ERC).
2. Biosynthesis: proteins are transported from ER to
the Golgi complex from where they are delivered to
the cell surface in secretory vesicles (SV)
Ran family
 Ran proteins: in every nucleated cell of every eucaryotic organism – functions:
• regulation of the active transport between the nucleus and cytoplasm
(TFs, histons from the cytoplasm to the nucleus, tRNA and mRNA vice versa)
 RanGAP is in the cytoplasm  RanGTP is converted here to RanGDP
 RanGEF is in the nucleus  RanGDP is converted here to RanGTP
 transport from the cytoplasm to the nucleus: RanGTP in the nucleus binds
the import complexes and releases the cargo
 transport from the nucleus to the cytoplasm: RanGTP in the nucleus
supports the formation of the export complexes → hydrolysis of GTP in
the cytoplasm (RanGAP) → release of the cargo in the cytoplasm
• mitotic spindle assembly and re-assembly of the nuclear envelope during
mitosis (RanGTP releases the proteins necessary for spindle assembly)
Rho family
 Actin cytoskeleton regulators  modulate:
•
•
•
•
•
cell migration
cell shape
vesicular trafficking, secretion
proliferation and transformation (the mechanism is still not known)
cell-cell and cell-matrix interactions
Effectors of the Rho family
Rho GTPase
Effectors
Function
RhoA, B, C
ROCK I, II (Rhoactivated kinase)
actomyosin contractility
cell adhesion
Cdc42
WASP
actin polymerization and filopodia
formation…essential for directionality
Rac1, 2, 3
IRSp53
actin polymerization and lamellipodia
formation…driving force in cell migration
A model of effector activation by Rho GTPases
Cdc42-induced WASP activation promotes filopodia formation
•
•
In the absence of Cdc42-GTP, WASP is in an auto-inhibited conformation
Upon Cdc42 activation by GTP binding, WASP binds to Cdc42-GTP, which is accompanied
by a conformational change exposing WASP VCA domain. This domain is now available to
activate Arp2/3 complex, stimulating actin polymerization.