This entry represents a group of BTB/POZ domain-containing proteins, such as BACURD1-3 from humans. They act as the substrate-specific adapters of a BCR (BTB-CUL3-RBX1) E3 ubiquitin-protein ligase complex, which mediates the ubiquitination of RhoA, leading to its degradation by the proteasome []. This entry also includes EAP3 from Arabidopsis. The BTB/POZ domain of EAP3 displays poor conservation of the residues required for CUL3 binding and is not likely to function as an E3 ligase adaptor [].
ARHGEF17 is a guanine nucleotide exchange for for RhoA GTPases; it contains a Dbl homology (DH) domain but lacks the typical pleckstrin homology domain [].
ARHGEF2, also called GEF-H1, acts as guanine nucleotide exchange factor (GEF) for RhoA GTPases []. It is thought to play a role in actin cytoskeleton reorganization in different tissues since its activation induces formation of actin stress fibres. ARHGEF2 contains a C1 domain followed by Dbl-homology (DH) and pleckstrin-homology (PH) domains which bind and catalyze the exchange of GDP for GTP on RhoA []. This entry represents the PH domain of ARHGEF2.
Rho family-interacting cell polarization regulator 2 (also known as protein FAM65B) is a plasma membrane-associated protein of hair cell stereocilia that is essential for hearing []. It is important for formation of the HDAC6-dysferlin protein complex during myogenic cell differentiation []. It is a RhoA inhibitor involved in T lymphocyte migration and neutrophil polarization [, ].
This entry includes pleckstrin homology domain-containing family G member 5/7 (PKHG5/7). PKHG5 is a guanine nucleotide exchange factor. PKHG5, also known as Plekhg5, has been shown to activate RHOA []. Plekhg5-depleted cultured motoneurons show defective axon growth and impaired autophagy of synaptic vesicles, which can be rescued by constitutively active Rab26 []. The function of PKHG7 is not clear.
Cyclin-dependent kinase inhibitor 1B (CDKN1B, also known as p27Kip1) controls cell proliferation by binding to and regulating the activity of cyclin-dependent kinases (Cdks) []. It is also involved in cell migration through binding and regulation of RhoA or stathmin [, , ]. It also inhibits DNA replication by binding to the DNA replication licensing factor MCM7 [].
Rho GTPase-activating protein 6 (RhoGAP6, also known as ARHGAP6) is both a GTPase-activating protein (GAP) with specificity for RhoA and a cytoskeletal protein that promotes actinremodelling []. It binds to and upregulates phospholipase C-delta, indicating a role for ARHGAP6 in enhanced activity of phospholipase C in hypertension [].
ARHGEF28 (also called RIP2 or p190RhoGEF) belongs to regulator of G-protein signaling (RGS) domain-containing RhoGEFs that are RhoA-selective and directly activated by the Galpha12/13 family of heterotrimeric G proteins. In addition to the Dbl homology (DH)-PH domain, p190RhoGEF contains an N-terminal C1 (Protein kinase C conserved region 1) domain. The DH-PH domains bind and catalyze the exchange of GDP for GTP on RhoA [].
ARHGEF12 (also called LARG) is a Rho guanine-nucleotide exchange factor that are RhoA-selective and directly activated by the Galpha12/13 family of heterotrimeric G proteins. LARG contains a regulator of G protein signaling (RGS) homology (RH) domain, the catalytic Dbl homology (DH) domain and the pleckstrin homology (PH) domain. The DH and PH domains bind RhoA and catalyze the exchange of GDP for GTP on RhoA. The active site of RhoA adopts two distinct GDP-excluding conformations among the four unique complexes in the asymmetric unit. The LARG PH domain also contains a potential protein-docking site. LARG forms a homotetramer via its DH domains []. This entry represents the PH domain of ARHGEF12. It has an exposed hydrophobic patch that could interface with other domains of LARG or other regulatory proteins [].
This entry represents the PH domain of guanine nucleotide exchange factor DBS. The DBS PH domain participates in binding to both the Cdc42 and RhoA GTPases []. PH domains have diverse functions, but in general are involved in targeting proteins to the appropriate cellular location or in the interaction with a binding partner [].DBS, also called MCF2L or OST, functions as a Rho GTPase guanine nucleotide exchange factor (RhoGEF), facilitating the exchange of GDP and GTP. It was originally isolated from a cDNA screen for sequences that cause malignant growth. It plays roles in regulating clathrin-mediated endocytosis and cell migration through its activation of Rac1 and Cdc42 [, ]. Depending on cell type, DBS can also activate RhoA and RhoG [, ]. DBS contains a Sec14-like domain [], spectrin-like repeats, a RhoGEF or Dbl homology (DH) domain, a Pleckstrin homology (PH) domain [], and an SH3 domain.
Anillin is a protein involved in septin organisation during cell division. It is an actin binding protein that is localised to the cleavage furrow, and it maintains the localisation of active myosin, which ensures the spatial control of concerted contraction during cytokinesis [].This entry represents a conserved domain found in anillin and anillin-like proteins. This domain shares homology with the RhoA binding protein Rhotekin [].
This entry includes Rho guanine nucleotide exchange factors 10 and 17.ARHGEF10 is a Rho guanine nucleotide exchange factor that may play a role in developmental myelination of peripheral nerves []. It was found to regulate mitotic spindle formation and play a role in the regulation of the cell division cycle [].ARHGEF17 is a guanine nucleotide exchange for for RhoA GTPases; it contains a Dbl homology (DH) domain but lacks the typical pleckstrin homology domain [].
Myosin phosphatase-RhoA interacting protein (M-RIP) is proposed to play a role in myosin phosphatase regulation by RhoA. M-RIP contains two PH domains followed by a Rho binding domain (Rho-BD), and a C-terminal myosin binding subunit (MBS) binding domain (MBS-BD). The amino terminus of M-RIP with its adjacent PH domains and polyproline motifs mediates binding to both actin and Galpha. M-RIP brings RhoA and MBS into close proximity where M-RIP can target RhoA to the myosin phosphatase complex to regulate the myosin phosphorylation state. M-RIP does this via its C-terminal coiled-coil domain which interacts with the MBS leucine zipper domain of myosin phosphatase, while its Rho-BD, directly binds RhoA in a nucleotide-independent manner [, ].PH domains have diverse functions, but in general are involved in targeting proteins to the appropriate cellular location or in the interaction with a binding partner []. They share little sequence conservation, but all have a common fold, which is electrostatically polarized. Less than 10% of PH domains bind phosphoinositide phosphates (PIPs) with high affinity and specificity []. PH domains are distinguished from other PIP-binding domains by their specific high-affinity binding to PIPs with two vicinal phosphate groups: PtdIns(3,4)P2, PtdIns(4,5)P2 or PtdIns(3,4,5)P3 which results in targeting some PH domain proteins to the plasma membrane []. A few display strong specificity in lipid binding. Any specificity is usually determined by loop regions or insertions in the N terminus of the domain, which are not conserved across all PH domains. PH domains are found in cellular signaling proteins such as serine/threonine kinase, tyrosine kinases, regulators of G-proteins, endocytotic GTPases, adaptors, as well as cytoskeletal associated molecules and in lipid associated enzymes [].
HGAL (human germinal centre-associated lymphoma, also known as a germinal centre-expressed transcript 2 or germinal centre-associated signalling and motility protein) is part of a family of mammalian sequences typically between 104 and 179 amino acids in length. Members were discovered in a search for proteins precipitating diffuse large B-cell lymphomas [, ]. HGAL and its murine homologue, M17 protein, are specifically expressed in germinal centre (GC) B-lymphocytes [].HGAL interacts with the cytoskeleton and aids the activity of interleukin-6 on cell migration []. It also modulates the RhoA signalling pathway []. HGAL protein also regulates B-cell receptor (BCR) signalling []. It seems that membrane-bound and cytoplasmic HGAL regulates distinct biological processes in B-cells.
A-kinase anchor protein 13 (AKAP13, also known as AKAP-Lbc) belongs to the AKAP family, which contains functionally related proteins involved in the targeting of the PKA holoenzyme towards specific physiological substrates []. AKAP13 anchors cAMP-dependent protein kinase (PKA) and acts as a guanine nucleotide exchange factor (GEF) that activates the GTPase RhoA []. The Rho-GEF activity of AKAP13 can be inhibited by protein 14-3-3 when it adopts an oligomeric conformation []. AKAP13 has been shown to be essential for cardiac development in mice []. It also plays a role in osteogenesis [].
VANGL proteins play important roles in the establishment of planar cell polarity (PCP) []. Vangl2 is required for retinal axon guidance [], kidney-branching morphogenesis and glomerular maturation []. It also plays a role in the orientation of stereociliary bundles in the cochlea and is required for polarization and movement of myocardializing cells in the outflow tract and seems to act via RHOA signaling to regulate this process []. It is required for cell surface localization of FZD3 and FZD6 in the inner ear [].
Rho GTPase-activating protein 26 (ARHGAP26), also known as GTPase regulator associated with focal adhesion kinase (GRAF), is a GTPase-activating protein for the small GTPases of the Rho family RhoA and CDC42 [, ]. GRAF influences cytoskeletal changes mediated by Rho proteins []. It is recognised as a tumor suppressor []. GRAF contains an N-terminal BAR domain, followed by a Pleckstrin homology (PH) domain, a Rho GAP domain, and a C-terminal SH3 domain. The SH3 domain of GRAF binds PKNbeta, a target of the small GTPase Rho [].
This entry represents Tax1-binding protein 3, also known as TIP-1, which is a PDZ domain-containing protein that functions in a wide variety of biological events through selective interaction with different proteins. It interacts with the RhoA effector protein rhotekin []. It also associates with glutaminase L [], potassium channel Kir2.3 []and NMDA receptors []. In humans, Tax1-binding protein 3 has been shown to be involved in the activation of Cdc42 by the viral protein HPV16 E6 []. TIP-1 can have both an oncogenic function or act as a tumor suppressor, probably by modulating the transcriptional activity of beta-catenin [, ].
Rho GTPase-activating proteins (RhoGAPs or ARHGAPs) bind to Rho proteins and enhance the hydrolysis rates of bound GTP. ARHGAP9 functions as a GAP for Rac and Cdc42, but not for RhoA []. It negatively regulates cell migration and adhesion []. It also acts as a docking protein for the MAP kinases Erk2 and p38alpha, and may facilitate cross-talk between the Rho GTPase and MAPK pathways to control actin remodeling []. It contains SH3, WW, Pleckstin homology (PH), and RhoGAP domains. This entry represents the SH3 domain found in ARHGAP9.
This entry represents the SH3 domain of GRAF.Rho GTPase-activating protein 26 (ARHGAP26), also known as GTPase regulator associated with focal adhesion kinase (GRAF), is a GTPase-activating protein for the small GTPases of the Rho family RhoA and CDC42 [, ]. GRAF influences cytoskeletal changes mediated by Rho proteins []. It is recognised as a tumor suppressor []. GRAF contains an N-terminal BAR domain, followed by a Pleckstrin homology (PH) domain, a Rho GAP domain, and a C-terminal SH3 domain. The SH3 domain of GRAF binds PKNbeta, a target of the small GTPase Rho [].
GRAF2, also called Rho GTPase activating protein 10 (ARHGAP10) or PS-GAP, is a GAP with activity towards Cdc42 and RhoA []. It regulates caspase-activated p21-activated protein kinase-2 (PAK-2p34). GRAF2 interacts with PAK-2p34, leading to its stabilization and decrease of cell death []. It is highly expressed in skeletal muscle, and is involved in alpha-catenin recruitment at cell-cell junctions []. GRAF2 contains an N-terminal BAR domain, followed by a Pleckstrin homology (PH) domain, a Rho GAP domain, and a C-terminal SH3 domain. The SH3 domain of GRAF binds PKNbeta, a target of the small GTPase Rho []. This entry represents the SH3 domain of GRAF2.
This entry represents the SH3 domain of DBS.DBS, also called MCF2L or OST, functions as a Rho GTPase guanine nucleotide exchange factor (RhoGEF), facilitating the exchange of GDP and GTP. It was originally isolated from a cDNA screen for sequences that cause malignant growth. It plays roles in regulating clathrin-mediated endocytosis and cell migration through its activation of Rac1 and Cdc42 [, ]. Depending on cell type, DBS can also activate RhoA and RhoG [, ]. DBS contains a Sec14-like domain [], spectrin-like repeats, a RhoGEF or Dbl homology (DH) domain, a Pleckstrin homology (PH) domain [], and an SH3 domain.
Net1 (also called ArhGEF8) is a RhoA-specific GEF (guanine nucleotide exchange factors). Net1 binds to caspase activation and recruitment domain (CARD)- and membrane-associated guanylate kinase-like domain-containing (CARMA) proteins and regulates nuclear factor kB activation []. In nucleus, it also regulates RhoB-mediated cell death after DNA damage []. Net1 and RhoA have been shown to play important roles in various aspects of vertebrate embryonic development and organogenesis [].Net1 contains a catalytic Dbl homology (DH) domain and an adjacent pleckstrin homology (PH) domain flanked by N- and C-terminal extensions []. This entry represents the PH domain. The DH and PH domains are necessary for binding to the GTPase and stimulating nucleotide exchange activity [].
GRAF2, also called Rho GTPase activating protein 10 (ARHGAP10) or PS-GAP, is a GAP with activity towards Cdc42 and RhoA []. It regulates caspase-activated p21-activated protein kinase-2 (PAK-2p34). GRAF2 interacts with PAK-2p34, leading to its stabilization and decrease of cell death []. It is highly expressed in skeletal muscle, and is involved in alpha-catenin recruitment at cell-cell junctions []. GRAF2 contains an N-terminal BAR domain, followed by a Pleckstrin homology (PH) domain, a Rho GAP domain, and a C-terminal SH3 domain. The SH3 domain of GRAF binds PKNbeta, a target of the small GTPase Rho [].
The Rnd proteins, which form a distinct sub-group of the Rho family of small GTP-binding proteins, have been shown to regulate the organization of the actin cytoskeleton in several tissues []. RhoN (also known as Rnd2 or Rho7) is a member of the novel Rho subfamily Rnd, together with Rnd1/Rho6 and Rnd3/RhoE/Rho8. Unlike other small G proteins, Rnd2/RhoN, Rnd3/RhoE, and Rnd1/Rho6 and do not hydrolyze GTP []. This is due to changes in key amino acids involved in catalysing GTP hydrolysis [].Rnd2/RhoN is transiently expressed in radially migrating cells in the brain while they are within the subventricular zone of the hippocampus and cerebral cortex. These migrating cells typically develop into pyramidal neurons. Cells that exogenously expressed Rnd2/RhoN failed to migrate to upper layers of the brain, suggesting that Rnd2/RhoN plays a role in the radial migration and morphological changes of developing pyramidal neurons, and that Rnd2/RhoN degradation is necessary for proper cellular migration. The Rnd2/RhoN GEF Rapostlin is found primarily in the brain and together with Rnd2/RhoN induces dendrite branching [, ]. Unlike Rnd1/Rho6 and Rnd3/RhoE/Rho8, which are RhoA antagonists, Rnd2/RhoN binds the GEF Pragmin and significantly stimulates RhoA activity and Rho-A mediated cell contraction []. Rnd2/RhoN is also found to be expressed in spermatocytes and early spermatids, with male-germ-cell Rac GTPase-activating protein (MgcRacGAP), where it localizes to the Golgi-derived pro-acrosomal vesicle [].
This entry is composed of Memo 1 (mediator of ErbB2-driven cell motility 1) and related proteins from eukaryotes, archaea and bacteria whose molecular function is unclear.Memo 1 is an effector of the ErbB2 receptor tyrosine kinase involved in breast carcinoma cell migration []. Its increased expression is associated with cancer aggressiveness. In breast cancer it regulates insulin-like growth factor-I receptor-dependent signaling pathway []. It binds to a specific ErbB2-derived phosphopeptide []. It regulates the localisation of the small G protein RhoA and its effector mDia1 at the plasma membrane, and thereby coordinates the organisation of the lamellipodial actin network, adhesion site formation, and MT outgrowth within the cell leading edge to sustain cell motility []. In yeast, the homologue is known as Mho1, and inhibits haploid invasive growth when overexpressed [].
Epithelial Cell Transforming Sequence 2 (Ect2) is a guanine nucleotide exchange factor (GEF) that catalyses the exchange of GDP for GTP. Like other GEFs, Ect2 serves as molecular switches in diverse signaling pathways, including cell polarity, mitotic spindle assembly, cytokinesis, etc [, , ]. Ect2 interacts with several members of the Rho GTPase family including, RhoA, RhoB, RhoC, RhoG, Rac1 and Cdc42 [, , , , ]. Ect2 regulates the formation of the actomyosin contractile ring at mitotic exit through activation of RhoA and functions in metaphase for the process of spindle assembly through activation of Cdc42 []. Ect2 is regulated through a number of mechanisms including phosphorylation, intracellular localization and intra- and inter-molecular interactions []. Ect2 is established as a human oncogene []. It is highly expressed in a variety of human tumors including brain lung, bladder, esophageal, pancreatic and ovarian tumors [, , , , ]. Ect2 is also suggested to play a role in neuronal differentiation and brain development [].
HR1 was first described as a three times repeated homology region of the N-terminal non-catalytic part of protein kinase PRK1(PKN) []. The first two of these repeats were later shown to bind the small G protein rho [, ]known to activate PKN in its GTP-bound form. Similar rho-binding domains also occur in a number of other protein kinases and in the rho-binding proteins rhophilin and rhotekin. Recently, the structure of the N-terminal HR1 repeat complexed with RhoA has been determined by X-ray crystallography. This domain contains two long alpha helices forming a left-handed antiparallel coiled-coil fold termed the antiparallel coiled- coil (ACC) finger domain. The two long helices encompass the basic region and the leucine repeat region, which are identified as the Rho-binding region [, , ].
This family consists of Rap1 GTPase-GDP dissociation stimulator 1 (RAP1GDS1, SmgGDS) from vertebrates and its homologues from fungi and Drosophila, GTPase-GDP dissociation stimulator vimar. RAP1GDS1 acts as a guanine exchange factor (GEFs) acting on small GTPases RhoA and RhoC through a distinct exchange mechanism compared with canonical GEFs []. RAP1GDS1 promotes both cell proliferation and migration and is up-regulated in several types of cancer [, ]. RAP1GDS1 plays a role in mitochondria morphology, as it regulates mitochondrial dynamics by controlling RHOT function to promote mitochondrial fission during high calcium conditions []. The Saccharomyces cerevisiae orthologue is known as Bem4. Bem4 interacts with Rho-type GTPases, including Cdc42p, and regulates the filamentous-growth mitogen-activated protein kinase pathway [, ].
Ephexin-1 is a RhoGEF (Rho-type guanine nucleotide exchange factor) that activates RhoA, Tac1, and Cdc42 by exchanging bound GDP for free GTP []. It is expressed mainly in the brain in a region associated with movement control. It regulates the stability of postsynaptic acetylcholine receptor (AChR) clusters and thus plays a critical role in the maturation and neurotransmission of neuromuscular junctions []. Ephexin-1 directly interacts with the ephrin receptor EphA4 and their coexpression enhances the ability of ephexin-1 to activate RhoA []. It is required for normal axon growth and EphA-induced growth cone collapse []. Ephexin-1 contains RhoGEF (also called Dbl-homologous or DH), Pleckstrin Homology (PH) and SH3 domains. This entry represents the SH3 domain.
Rho GTPase-activating protein 26 (ARHGAP26), also known as GTPase regulator associated with focal adhesion kinase (GRAF), is a GTPase-activating protein for the small GTPases of the Rho family RhoA and CDC42 [, ]. GRAF influences cytoskeletal changes mediated by Rho proteins []. It is recognised as a tumor suppressor []. GRAF contains an N-terminal BAR domain, followed by a Pleckstrin homology (PH) domain, a Rho GAP domain, and a C-terminal SH3 domain. The SH3 domain of GRAF binds PKNbeta, a target of the small GTPase Rho [].This entry represents the BAR domain of GRAF. BAR domains form dimers that bind to membranes, induce membrane bending and curvature, and may also be involved in protein-protein interactions. The BAR domain of GRAF directly interacts with its Rho GAP domain and inhibits its activity. Autoinhibited GRAF is capable of binding membranes and tubulating liposomes, showing that the membrane-tubulation and GAP-inhibitory functions of the BAR domain can occur simultaneously [].
LAMTOR1 is a family of eukaryotic proteins that have otherwise been referred to as Lipid raft adaptor protein p18, Late endosomal/lysosomal adaptor and MAPK and MTOR activator 1, and Protein associated with DRMs and endosomes.LAMTOR1 regulates the mTOR (mammalian target of rapamycin) pathway, a signaling cascade that promotes cell growth in response to growth factors, energy levels, and amino acids. LAMTOR1 is part of the Ragulator complex, recruits the Rag GTPases and the mTORC1 complex to lysosomes, a key step in activation of the TOR signaling cascade by amino acids []. LAMTOR1 is responsible for anchoring the Ragulator complex to membranes. It may regulate both the recycling of receptors through endosomes and the MAPK signaling pathway through recruitment of some of its components to late endosomes []. It may be involved in cholesterol homeostasis regulating LDL uptake and cholesterol release from late endosomes/lysosomes. It may also play a role in RHOA activation [, ].This family also includes Saccharomyces cerevisiae MEH1 (Ego1), a component of the EGO complex. The EGO complex and the Rag-Ragulator complex are structurally related []. The yeast EGO complex consists of Gtr1, Gtr2, Ego1, and Ego3, localises to the endosomal and vacuolar membranes, and plays a crucial role in cell growth and autophagy regulation through amino acid signals that activate TORC1 []. MEH1 (GSE2) is also a component of the GSE complex, which is required for proper sorting of amino acid permease Gap1 [].
Toxins A (TcdA) and B (TcdB) of Clostridium difficile belong to the family of clostridial glucosylating toxins. These toxins glucosylate small GTPases of Rho and Ras families, inhibiting the signalling and regulatory functions of these switch proteins. After receptor-binding, the toxins are endocytosed to reach acidic endosomal compartments from where the toxins are translocated into the cytosol []. TcdB has been shown to consist of a N-terminal glucosyltransferase domain (GTD), responsible for the biological effects of the toxin, a cysteine protease domain (CPD), responsible for autocatalytic cleavage, a hydrophobic region (HR), which has been suggested to be involved in toxin translocation, and a C-terminal repetitive domain involved in receptor binding. The pore-forming region of toxin B has been described to be in a region in the middle of the protein, within amino acid residues 830 and 990 [].This entry represents the N-terminal glucosyltransferase domain from TcdA and TcdB. It is also found in other toxins. The GTD of TcdB has been shown to glycosylate the host's RhoA protein [].
PH domains have diverse functions, but in general are involved in targeting proteins to the appropriate cellular location or in the interaction with a binding partner []. They share little sequence conservation, but all have a common fold, which is electrostatically polarized. Less than 10% of PH domains bind phosphoinositide phosphates (PIPs) with high affinity and specificity []. PH domains are distinguished from other PIP-binding domains by their specific high-affinity binding to PIPs with two vicinal phosphate groups: PtdIns(3,4)P2, PtdIns(4,5)P2 or PtdIns(3,4,5)P3 which results in targeting some PH domain proteins to the plasma membrane []. A few display strong specificity in lipid binding. Any specificity is usually determined by loop regions or insertions in the N terminus of the domain, which are not conserved across all PH domains. PH domains are found in cellular signaling proteins such as serine/threonine kinase, tyrosine kinases, regulators of G-proteins, endocytotic GTPases, adaptors, as well as cytoskeletal associated molecules and in lipid associated enzymes [].ARHGEF3 (also known as XPLN) is a guanine nucleotide exchange factor (GEF) for RhoA and RhoB GTPases []. It contains a tandem Dbl homology and a PH domain. This entry represents the PH domain.
HR1 was first described as a three times repeated homology region of the N-terminal non-catalytic part of protein kinase PRK1(PKN) []. The first two of these repeats were later shownto bind the small G protein rho [, ]known to activate PKN in its GTP-bound form. Similar rho-binding domains also occur in a number of other protein kinases and in the rho-binding proteins rhophilin and rhotekin. Recently, the structure of the N-terminal HR1 repeat complexed with RhoA has been determined by X-ray crystallography. This domain contains two long alpha helices forming a left-handed antiparallel coiled-coil fold termed the antiparallel coiled- coil (ACC) finger domain. The two long helices encompass the basic region and the leucine repeat region, which are identified as the Rho-binding region [, , ].This entry also includes Transducer of Cdc42-dependent actin assembly protein (TOCA) family proteins which contains a central HR1 (also known as Rho effector motif class 1, REM-1) which is closely related to Cdc42-interacting protein 4 (CIP4), effectors of the Rho family small G protein Cdc2 [].
AMPK, a serine/threonine protein kinase (STK), catalyzes the transfer of the gamma-phosphoryl group from ATP to S/T residues on protein substrates. It acts as a sensor for the energy status of the cell and is activated by cellular stresses that lead to ATP depletion such as hypoxia, heat shock, and glucose deprivation, among others. AMPK is a heterotrimer of three subunits: alpha, beta, and gamma []. The alpha subunit is the catalytic subunit and it contains an N-terminal kinase domain, a putative autoinhibitory domain (AID) and a C-terminal region required for beta subunit binding. The beta scaffolding subunit mediates AMPK assembly by bridging alpha and gamma subunits. The C-terminal domain of the AMPK alpha 1 subunit interacts with the C-terminal region of the beta subunit to form a tightalpha-beta complex that is associated with the gamma subunit. The AMPK alpha subunit auto-inhibitory region interacts with the kinase domain; this inhibition is negated by the interaction with the AMPK gamma subunit [].AMPK has been implicated in a number of diseases related to energy metabolism including type 2 diabetes, obesity and cancer [, ]. AMPK is activated by rising AMP concentrations coupled with falling ATP concentrations. Activation of AMPK is also dependent on the phosphorylation of alpha subunit by upstream kinases such as LKB1 [].Vertebrates contain two isoforms of the alpha subunit, alpha1 and alpha2, which are encoded by different genes, PRKAA1 and PRKAA2, respectively, and show varying expression patterns. AMPKalpha1 is the predominant isoform expressed in bone; it plays a role in bone remodeling in response to hormonal regulation []. AMPK alpha1 impacts the regulation of fat metabolism []. It also mediates the vasoprotective effects of estrogen through phosphorylation of another in vivo substrate, RhoA [].
Small GTPases form an independent superfamily within the larger class of regulatory GTP hydrolases. This superfamily contains proteins that control a vast number of important processes and possess a common, structurally preserved GTP-binding domain [, ]. Sequence comparisons of small G proteins from various species have revealed that they are conserved in primary structures at the level of 30-55% similarity [].Crystallographic analysis of various small G proteins revealed the presence of a 20kDa catalytic domain that is unique for the whole superfamily [, ]. The domain is built of five alpha helices (A1-A5),six β-strands (B1-B6) and five polypeptide loops (G1-G5). A structural comparison of the GTP- and GDP-bound form, allows one to distinguish two functional loop regions: switch I and switch II that surround the gamma-phosphate group of the nucleotide. The G1 loop (also called the P-loop) that connects the B1 strand and the A1 helix is responsible for the binding of the phosphate groups. The G3 loop provides residues for Mg2 and phosphate binding and is located at the N terminus of the A2 helix. The G1 and G3 loops are sequentially similar to Walker A and Walker B boxes that are found in other nucleotide binding motifs. The G2 loop connects the A1 helix and the B2 strand and contains a conserved Thr residue responsible for Mg2 binding. The guanine base is recognised by the G4 and G5 loops. The consensus sequence NKXD of the G4 loop contains Lys and Asp residues directly interacting with the nucleotide. Part of the G5 loop located between B6 and A5 acts as a recognition site for the guanine base [].The small GTPase superfamily can be divided into at least 8 different families, including:Arf small GTPases. GTP-binding proteins involved in protein trafficking by modulating vesicle budding and uncoating within the Golgi apparatus.Ran small GTPases. GTP-binding proteins involved in nucleocytoplasmic transport. Required for the import of proteins into the nucleus and also for RNA export.Rab small GTPases. GTP-binding proteins involved in vesicular traffic.Rho small GTPases. GTP-binding proteins that control cytoskeleton reorganisation.Ras small GTPases. GTP-binding proteins involved in signalling pathways.Sar1 small GTPases. Small GTPase component of the coat protein complex II (COPII) which promotes the formation of transport vesicles from the endoplasmic reticulum (ER).Mitochondrial Rho (Miro). Small GTPase domain found in mitochondrial proteins involved in mitochondrial trafficking.Roc small GTPases domain. Small GTPase domain always found associated with the COR domain.This entry represents the Rho subfamily of Ras-like small GTPases. The small GTPase-like protein LIP2 (light insensitive period 2) from Arabidopsis thalianais implicated in control of the plant circadian rhythm []. The crystal structures of a number of the members of this entry have been determined: Rnd3/RhoE [], RhoA []and Cdc42 [].
G protein-coupled receptors (GPCRs) constitute a vast protein family that encompasses a wide range of functions, including various autocrine, paracrine and endocrine processes. They show considerable diversity at the sequence level, on the basis of which they can be separated into distinct groups []. The term clan can be used to describe the GPCRs, as they embrace a group of families for which there are indications of evolutionary relationship, but between which there is no statistically significant similarity in sequence []. The currently known clan members include rhodopsin-like GPCRs (Class A, GPCRA), secretin-like GPCRs (Class B, GPCRB), metabotropic glutamate receptor family (Class C, GPCRC), fungal mating pheromone receptors (Class D, GPCRD), cAMP receptors (Class E, GPCRE) and frizzled/smoothened (Class F, GPCRF) [, , , , ]. GPCRs are major drug targets, and are consequently the subject of considerable research interest. It has been reported that the repertoire of GPCRs for endogenous ligands consists of approximately 400 receptors in humans and mice []. Most GPCRs are identified on the basis of their DNA sequences, rather than the ligand they bind, those that are unmatched to known natural ligands are designated by as orphan GPCRs, or unclassified GPCRs [].The rhodopsin-like GPCRs (GPCRA) represent a widespread protein family that includes hormone, neurotransmitter and light receptors, all of which transduce extracellular signals through interaction with guanine nucleotide-binding (G) proteins. Although their activating ligands vary widely in structure and character, the amino acid sequences of the receptors are very similar and are believed to adopt a common structural framework comprising 7 transmembrane (TM) helices [, , ].G2A is expressed mainly in lymphocytes and its expression is up-regulated by stress and prolonged mitogenic signals. Micelacking the receptor have been found to develop a late-onset autoimmunedisease []. It has therefore been suggested that G2A may function as a sensor of LPC levels at sites of inflammation and act as a negativeregulator of lymphocyte growth to limit expansion of tissue-infiltratingcells and overt autoimmune disease. Activation of G2A by LPC results inan increase in intracellular calcium levels (through coupling to Giproteins) and activation of MAP kinases. The receptor has also been shown to couple to G13 proteins, causing RhoA activation and formation of actinstress fibres.
