Monomeric G proteins mirka.rovenska@lfmotol.cuni.cz

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 = 20-25 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

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

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

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

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

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

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 http://uk.images.search.yahoo.com/search/images;_ylt=A0WTf2xcsHpLlWsAjaJNBQx.?p=sos+ras+sh2&fr=yfp-t-702&ei=utf-8&x=wrt&y=Search

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

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

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

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

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

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)

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…

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)

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)

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

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

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.