Monomeric G proteins Alice Skoumalová

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

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

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) 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

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

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

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

The G domain of small GTPases Obrázek

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

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)

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)

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)

The cellular localization of Rab GTPases

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

(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

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 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