Download Monomeric G proteins

Monomeric G proteins
Alice Skoumalová
1. General features of chemical messengers - types of receptors
2. Endocrine, paracrine and autocrine actions
3. Examples of chemical messengers
Ion-channel receptors
Protein kinase receptors
Heptahelical receptors (G protein-coupled receptors)
G proteins = GTP binding proteins
 They bind GTP (active conformation)
 They regulate a wide variety of processes in the cell
Heterotrimeric G proteins
• 3 subunits (α, β, γ)
• α-subunit binds GTP, dissociates and
interacts with an effector
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)
GTP
Small GTPases
• 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
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
Lipid post-translational modification (prenylation)
1. Farnesyltransferase (Ras): the covalent addition of a C15 farnesyl isoprenoid (F)
lipid to the cysteine residue of the CAAX motif
2. Geranylgeranyltransferase I (Rho): the covalent addition of a C-20 geranylgeranyl
isoprenoid (GG) lipid to the cysteine residue of the CAAX motif
3. Geranylgeranyltransferase II (Rab): the covalent addition of a C-20 geranylgeranyl
isoprenoid (GG) lipid to the cysteine residue of the CC, CXC, CCX, CCXXX
Guanine nucleotide exchange factors (GEFs)
•
•
regulate activation of small GTPases and their downstream effects
responsible for exchanging free cytosolic GTP for bound GDP
Diverse extracellular signals promote Ras activation by
stimulating GEFs
• common CDC25 homology domain, catalyzes nucleotide exchange
• different GEFs can be activated by different signals
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
Small GTPase structure
H-Ras functional domains
Obrázek
1. The G domain: four sequences involved in GTP-binding and GTPase activity (structurally
similar among small GTPases)
2. The CAAX motif: Ras and Rho
3. Core effector domain: effector binding
4. Switches I and II: change in conformation in the GDP- and GTP-proteins
5. RHO insert: additional sequences
Diverse functions of small GTPases
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: regulators of membrane trafficking (the secretory and endocytic
pahways)
Rho: regulators of actin cytoskeletal organization, which influences cell
morphology and movement
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)
Ras effectors
Ras proteins serve as signaling nodes
1. Various extracellular signals converge on Ras proteins:
Growth factors (epidermal growth factor, plateletderived 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)
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)
The cellular localization of Rab GTPases
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
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
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
Model of effector activation by Rho GTPases
Cdc42-induced WASP activation promotes filopodia
Questions
1. The principle of function of small GTPases, the cycle, regulations,
activations
2. The principle of lipid post-translational modification
3. The structure of small GTPases
4. The function of the Ras proteins, the results of Ras mutations in human
carcinoma
5. The function of the Rho and Rab proteins