1. Monomeric G proteins
Alice Skoumalová

2. G proteins = GTP binding proteins Heterotrimeric G proteins
They bind GTP (active conformation)
They regulate a wide variety of processes in the cell
Monomeric G proteins
= small G proteins or small GTPases
bind GTP (active GTP-bound conformation), interact with effectors, promote various cellular responses
catalyze the hydrolysis of the terminal phosphate group (inactive GDP-bound conformation)
Heterotrimeric G proteins
3 subunits (α, β, γ)
α-subunit binds GTP, dissociates and interacts with an effector
GTP

3. Small GTPases Regulatory proteins:
GDP/GTP-regulated molecular switches in signaling pathways („on“ and „off“)
two-state structural change that is fundamental for their biological function:
GTP-protein „on“
GDP-protein „off“
Regulatory proteins:
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): prevent nucleotide exchange

4. Classification of small GTPases
Over 150 mammalian proteins
The „Ras superfamily“ related proteins, further divided into families:
Ras, Rab, Rho, Arf, Rad, Ran, Rheb, Rit

6. Lipid post-translational modification (prenylation)
Farnesyltransferase (Ras): the covalent addition of a C15 farnesyl isoprenoid (F) lipid to the cysteine residue of the CAAX motif
Geranylgeranyltransferase I (Rho): the covalent addition of a C-20 geranylgeranyl isoprenoid (GG) lipid to the cysteine residue of the CAAX motif
Geranylgeranyltransferase II (Rab): the covalent addition of a C-20 geranylgeranyl isoprenoid (GG) lipid to the cysteine residue of the CC, CXC, CCX, CCXXX

7. Guanine nucleotide exchange factors (GEFs)
regulate activation of small GTPases and their downstream effects
responsible for exchanging free cytosolic GTP for bound GDP

8. Diverse extracellular signals promote Ras activation by stimulating GEFs
common CDC25 homology domain, catalyzes nucleotide exchange
different GEFs can be activated by different signals

9. GTPase activating proteins (GAPs)
accerelate the small GTPase hydrolysis
contain the GAP catalytic domain and additional sequences (involved in regulation)
The mutated Ras proteins found in human cancers - insensitive to GAP stimulation - are persistently GTP-bound - deregulated effector activation

10. Guanine nucleotide dissociation inhibitors (GDIs)
Two distinct negative regulatory functions:
1. bind and mask the isoprenoid lipid modification (prenylation)
2. inhibit GAP and GEF regulation

11. Small GTPase structure H-Ras functional domains
Obrázek
The G domain: four sequences involved in GTP-binding and GTPase activity (structurally similar among small GTPases)
The CAAX motif: Ras and Rho
Core effector domain: effector binding
Switches I and II: change in conformation in the GDP- and GTP-proteins
RHO insert: additional sequences

12. The G domain of small GTPases
Obrázek

13. Diverse functions of small GTPases Overview of small GTPase functions
Various cellular functions:
1. Distinct GAPs and GEFs are regulated by different input signals
2. Various effectors resulting in many different cellular responses
Overview of small GTPase functions
Ras: regulators of cell proliferation and differentiation
Rab, Arf, Sar: regulators of membrane trafficking (the secretory and endocytic pahways)
Ran: regulators of nucleocytoplasmic transport
Rho: regulators of actin cytoskeletal organization, which influences cell morphology and movement

14. Function: cell proliferation and differentiation
Ras GTPases
Function: cell proliferation and differentiation
The frequent mutations of Ras proteins in human cancers
Mutations that block GAP-stimulated GTP hydrolysis result in Ras proteins being locked in their active GTP-bound state (in various human tumors)

15. Ras proteins serve as signaling nodes
Ras effectors
Ras proteins serve as signaling nodes
1. Various extracellular signals converge on Ras proteins:
Growth factors (epidermal growth factor, platelet- derived growth factor)
Hormones (insulin)
Cytokines (interleukin-1)
The extracellular matrix proteins (via integrins)
2. Ras proteins regulate the activities of various effectors with divergent biochemical functions:
The Raf serine/threonine kinases (activate the Raf/Mek/Erk kinase cascade – the growth-regulatory functions)
The phosphatidylinositol 3-kinases (generate PIP3 – regulates various cascades)
Phospholipase Cε (generate DAG and Ca2+)
GEFs of other small GTPases (small GTPase cascades – Ral, Rac, Rab)

16. Function: membrane trafficking
Rab GTPases
(Ras homologue from brain)
Function: membrane trafficking
the largest family of small GTPases (at least 60 members)
localizing to specific organelles of the secretory and endocytic pathway
ubiquitously expressed; but cell-type specific Rab3a (expressed in neurons) and Rab27 (expressed in melanocytes and platelets)

17. The cellular localization of Rab GTPases

18. Mutation in Rab genes-membrane trafficking alterations-diseases
Rabs and diseases
Mutation in Rab genes-membrane trafficking alterations-diseases
Griscelli syndrome (an autosomal recessive disorder)
Pigmentation defects, immunological defects, neurological dysfunction
Mutations in the Rab27a gene (regulates the transport of melanosomes to the periphery of melanocytes and the secretion of lytic granules in cytotoxic T lymphocytes) result in the loss of pigmentation and in the uncontrolled activation of T lymphocytes
Choroideremia (an X-linked disease)
Degeneration of the pigment epithelium of the eye
Defect in the REP-1 gene (geranylgeranyltransferase that is essential for the prenylation of Rab27a in the retinal pigment epithelium) results in deficient melanosome transport in the pigment epithelium and lack of protection during light exposure

19. (Ras related nuclear protein)
Ran GTPases
(Ras related nuclear protein)
in every nucleated cell; essential for the viability of the cells
Function:
Regulate the active transport of cargo between the nucleus and cytoplasm
Essential for mitotic spindle assembly and placement, re-assembly of the nuclear envelope

20. Rho GTPases
Function: Regulation of actin cytoskeletal organization (cell morphology and movement)
Modulation of various cellular processes:
1. Cell migration: regulation of the actin cytoskeleton, coordinated activation of Rho proteins
2. Secretion and endocytosis: vesicle trafficking
3. Proliferation and transformation: invasiveness of cancer cells

21. Rho GTPase Effectors Function in cell biology
RhoA, B, C ROCK I, II Actomyosin contractility,
cell migration
Rac1, 2, 3 IRSp53 Actin polymerization, lamellipodia formation, driving force in cell migration
Cdc42 WASP/N-WASP Actin polymerization, filopodia formation, sensor role in cell migration

22. Model of effector activation by Rho GTPases
Cdc42-induced WASP activation promotes filopodia

23. Questions
The principle of function of small GTPases, the cycle, regulations, activations
The most important families of small GTPases
The principle of lipid post-translational modification
The results of Ras mutations in human carcinoma
The structure of small GTPases
The function of the Ras proteins
The function of the Rho and Rab proteins