1. Monomeric G proteins

2. 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
3 subunits (α,,γ)
activated by G protein-coupled receptors
GDP is exchanged for GTP in the α-subunit → α dissociates from γ and interacts with the effector; GTP is hydrolyzed → inactivation
Monomeric G proteins
= small GTPases: Mr = kDa
not directly activated by GPCRs
similarly to the α-subunit, GDP exchange for GTP leads to interaction with an effector; GTP is hydrolyzed → inactivation

3. Small GTPases
GDP/GTP-regulated molecular „switches“ in signalling pathways
Two-state structural change is fundamental for their biological function:
Effectors:
protein kinases
phosphatidylinositol 3-kinase: converts PIP2 to PIP3 which regulates the
activity of other target molecules
phospholipases…
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

4. 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

5. 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

6. Post-translational modification of small GTPases
by prenylation
Prenylation probably facilitates association of small GTPases with specific membrane compartments
Ras: farnesyltransferase – covalent addition of farnesyl isoprenoid (C15) to the Cys residue of the C-terminal CAAX motif (X = S, M)
Rho, Ras: geranylgeranyltransferase I – covalent addition of geranylgeranyl isoprenoid (C20) to the Cys residue of the C-terminal CAAX motif (X = L, F)
Rab: geranylgeranyltransferase II – covalent addition of geranylgeranyl isoprenoid to the Cys residues of the C-terminal motifs CC, CXC, CCX, or CCXXX

7. 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

8. 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

9. 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

10. 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

11. Small GTPase structure
All small GTPases share the G domain which contains: GTP-binding and hydroly-zing 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

12. 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

13. 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 phospho- rylation, it activates gene expression

14. 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)

15. 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…

16. 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)

17. 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)

18. 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

19. Effectors of the Rho family
Rho GTPase
Effectors
Function
RhoA, B, C
ROCK I, II (Rho-activated 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

20. 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.