Small G proteins

1
Small G proteins Chang Chung Chang.Chung_at_vand
erbilt.edu 468 RRB
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Small G proteins

• Small GTP-binding proteins are monomeric G
  proteins with molecular masses
• of 2030 kDa.

• Small G proteins act as a molecular
  switch, cycling between GDP (off) and GTP
  (on)-bound states.

• Small GTP-binding proteins function as
  molecular switches in fundamental events such as
  signal transduction, cytoskeleton dynamics and
  intracellular trafficking.

• Small G proteins are conserved in primary
  structures and are 30-55 homologous to each other

• More than 100 small G-proteins have been
  identified in eukaryotes from yeast to human

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Identification of Ras as an oncogene

• The Ha-Ras and Ki-Ras genes were first
  discovered as the v-Ha-Ras and v-Ki-Ras oncogenes
  of sarcoma viruses around 1980 and their cellular
  oncogenes were found in some human carcinomas in
  1982

• Since 1982, Ras has been the subject of
  intense research scrutiny, focused on determining
  the role of aberrant Ras function in human
  cancers and defining the mechanism by which Ras
  mediates its actions in normal and neoplastic
  cells.

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The members of small G-proteins are structurally
classified into at least five families the Ras,
Rho, Rab, Sar1/Arf, and Ran families
Gene Expression-proliferation
Actin Cytoskeleton
Vesicle Budding
Nuclearcytoplasmic transport
Vesicle Trafficking
Garcia-Ranea and Valencia (1998)
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Ras proteins act as a molecular switch, cycling
between GDP (off) and GTP (on)-bound states.

• Exchange of GTP for GDP is regulated by Guanidine
  nucleotide Exchange Factors, or GEFs.
• Binding of GTP causes a conformational change in
  the Ras molecule, especially in residues in the
  switch region. This creates new binding sites
  for effector molecules that in turn regulate
  downstream signaling pathways.
• GTP is hydrolyzed back to GDP by the intrinsic
  GTPase activity of Ras, which is enhanced several
  orders of magnitude by the action of
  GTPase-Activating Proteins, or GAPs.

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Consensus amino acid sequences responsible for
specific interaction with GDP and GTP and for
GTPase activity.
GAP Downstream effectors
12
17
40
Switch I
61
GDI
Switch II
Consensus sequence for GDP/GTP binding and GTPase
activity
cleaved

• Key residues
• Gly 12 Naturally occurring mutation to valine
  (12V) destroys the intrinsic GTPase activity and
  results in a constitutively active mutation. This
  mutation has been found in certain tumors.
• Gln 61 Mutation to leucine (61L) creates an
  activating mutation similar to 12V
• Thr 17 to asn (17N) Mutation that causes the Ras
  molecule to bind irreversibly to GEFs and remain
  inactive. This mutation is a dominant negative,
  presumably because the Ras proteins bind and
  sequester the GEFs, preventing them from
  activating native molecules.

Transfection of these constitutively active and
dominant negative isoforms represents a major
approach by which pathways and roles of small
GTPases have been defined.
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Crystal structure of Ras bound to GDP or the
non-hydrolyzable analog GppCp.
(Sprang, 1997).

• The effector loop is the region that binds to
  downstream effectors and is exposed upon GTP
  binding.
• The effector domain includes part of Switch I and
  Switch II regions, the regions that alter
  conformation following GTP binding. Specificity
  is generated by key residues within the effector
  loop, as determined by a set of mutations that
  uncouple specific effector pathways.

The positions of switch I and switch II (white in
free Cdc42), helix 1 and the -sheet (red) change
upon complex formation. Left, free Cdc42GMPPNP
right, Cdc42/fACK complex. The positions of
switches I and II in free Cdc42GMPPNP are
denoted by white lines, because they are flexible
and their positions cannot therefore be
determined.
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G-proteins are tightly regulated

• 3 types of accessory proteins that modulate
  cycling of G-proteins between GTP/GDP
• 1. GAPs GTPase-activating proteins. Stimulate
  GTP hydrolysis. Inactivate G-protein. Example of
  a GAP PLCb.
• 2. GEFs Guanine nucleotide-exchange factors
  G-protein-coupled receptors (GPCR). Stimulate
  dissociation of GDP (inactive) from G-protein so
  GTP can bind (active).
• 3. GDIs Guanine nucleotide-dissociation
  inhibitors. Inhibit release of bound GDP
  (maintain G-protein in inactive state).

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Exchange factors (GEFs) The link between
receptors and small G proteins

• Guanine nucleotide exchange factors (GEFs) are
  primarily responsible for linking cell-surface
  receptors to Ras protein activation by catalyzing
  the dissociation of GDP from the inactive Ras
  proteins.
• Most small G proteins bind guanine nucleotides
  very tightly
• Dissociation rate for Ras and Ran 10-5 S-1,
  Rho 10-4 S-1
• GEFs for the small GTPases are though to act by
  increasing the dissociation rate dramatically and
  stabilizing the nucleotide-free intermediate
  transition state, in a manner similar to that of
  GPCRs, which serve as GEFs for heterotrimeric G
  proteins.

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The guanine nucleotide exchange reaction
1
2
3
4
1. GEF forms a low affinity, docking complex with
GDP-bound Ras
2 GDP dissociates from the initial complex, then
it becomes high affinity, binary GEF-Ras
complex GEFs are also able to dissociate GTP,
but the dominant concentration of GTP and the
interaction of GTP-bound Ras and effectors drive
the reaction in the GDP to GTP direction
3 GTP binds to Ras
4 GEF is rapidly dissociated from Ras
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Exchange factors for Ras These molecules share
a Ras-GEF homology region. The best known members
of this family are Sos and RasGRF.
Glutamic finger
Activation of SOS
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Exchange factors for Rho proteins Dbl family The
dbl oncogene was identified in 1985 (Eva and
Aaronson) from B cell lymphoma. Dbl family
members have tandem dbl-homology (DH) and
pleckstrin-homology (PH) domains. The DH domain
is necessary for exchange activity, while the PH
domain helps target the molecule to the membrane.
DH without PH full GEF activity, no
transformation
13
Activation of Dbl
14

• GAP proteins
• catalyze the hydrolysis of GTP to GDP and ensure
  that Cdc42 cycles back to its starting or basal
  state.

• GAP introduces a conserved arginine residue
  (called the arginine finger) into the active
  site of Cdc42, which helps to catalyze GTP
  hydrolysis by stabilizing the transition state of
  the hydrolytic reaction.

Gly12 in the transition state mimic is within van
der Waals distance of both arginine-789 of GAP
and Gln61 of Ras, and even its mutation to
alanine would disturb the arrangements of
residues in the transition state.
Regions of RasGAP contacting Ras in light brown,
Ras in yellow, and GDP and AlF3 as ball-and-stick
models.
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• Lipid modification of small G proteins
• Ras proteins are lipid modified (by farnesyl or
  geranylgeranyl) at the C-terminal cysteine in the
  CAAX motif. In addition, those with a farnesyl
  group are also palmitoylated, with the exception
  of K-ras, which has a poly-basic region (a string
  of lysines).
• Rho proteins have a geranylgeranyl group without
  additional modification.
• These lipid modifications help to target small G
  proteins to the membrane.

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Regulation of Ras targeting
Ras proteins are synthesized initially as
cytosolic proteins in which farnesyltransferase
catalyses the covalent addition of the C15
farnesyl isoprenoid lipid. ER-associated enzymes
then catalse the proteolytic removal of the AAX
residues and carboxyl methylation of the
cysteine. These modifications alone are not
sufficient to traffick Ras to the plasma
membrane. Second signals, such as the
lysine-rich sequence of K-Ras or the addition of
palmitate to cysteine residues upstream of the
CAAX motif are also necessary.

• For Ras family members, both GDP and GTP bound
  forms are thought to reside at the membrane, but
  association into lipid microdomains is regulated
  by activation.

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Regulation of Rho targeting
For Rho family members, membrane targeting is
regulated by activation. The current model is
that GDIs (Guanidine Dissociation Inhibitors)
bind to the GDP-Rho via a hydrophobic pocket that
masks the lipid group. Upon activation displaced,
the lipid group is exposed, and Ras relocalizes
to the plasma membrane.
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Downstream signaling cascades and multiple
effectors of Ras
Elk-1
Biocarta.com

• Mutated versions of the three human Ras genes
  have been detected in 30 of all human cancers,
  implying an important role for aberrant Ras
  protein function in carcinogenesis

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Rho family G proteins

• Regulator of actin cytoskeleton
• Mediator of growth factor receptor effects

How was this paradigm determined? 1) C3 Botulinum
Toxin an ADP ribosyltransferase Irreversible ADP
ribosylation inhibits Rho function, inhibits
stress fibers 2) Constitutively active RhoV14
mimics LPA effects on stress fibers Dominant
negative Rho19N blocks 3) Constitutively active
RacV12 mimics PDGF effects on ruffles Dominant
negative Rac 17N blocks
(Takai et al. 2001)
Actin
Vinculin
(Hall 1988)
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Downstream signaling cascades of Rho family small
G proteins
Cytoskeletal Organization
Gene Expression
WAVE
(Takai et al. 2001)