cells Review Rho Family of Ras-Like GTPases in Early-Branching Animals 1,2, 3, 1, , Silvestar Beljan y, Maja Herak Bosnar y and Helena Cetkovi´c´ * y 1 Division of Molecular Biology, Ruder¯ Boškovi´cInstitute, HR-10000 Zagreb, Croatia; [email protected] 2 Division of Molecular Biology, Faculty of Science, University of Zagreb, HR-10000 Zagreb, Croatia 3 Division of Molecular Medicine, Ruder¯ Boškovi´cInstitute, HR-10000 Zagreb, Croatia; [email protected] * Correspondence: [email protected]; Tel.: +385-1-456-1115 These authors contributed equally to this work. y Received: 9 September 2020; Accepted: 9 October 2020; Published: 13 October 2020 Abstract: Non-bilaterian animals consist of four phyla; Porifera, Cnidaria, Ctenophora, and Placozoa. These early-diverging animals are crucial for understanding the evolution of the entire animal lineage. The Rho family of proteins make up a major branch of the Ras superfamily of small GTPases, which function as key molecular switches that play important roles in converting and amplifying external signals into cellular responses. This review represents a compilation of the current knowledge on Rho-family GTPases in non-bilaterian animals, the available experimental data about their biochemical characteristics and functions, as well as original bioinformatics analysis, in order to gain a general insight into the evolutionary history of Rho-family GTPases in simple animals. Keywords: non-bilaterian animals; Rho GTPases; Cdc24; Rho; Rac; RhoBTB; Miro; Porifera; Ctenophora; Placozoa; Cnidaria 1. Introduction The development of multicellular organisms depends on the ability of cells to detect and respond adequately to external signals, expressed by other cells. The intercellular signaling in embryonic development mediated by adhesion molecules, extracellular matrix, cytokines, morphogens, growth factors, or hormones has been extensively studied. Cell signaling is initiated by binding of ligands to their specific cell surface receptors, which are conversely converted into responses leading to gene transcription, cell shape modeling, adhesion, motility, and endo/exocytosis [1]. Although eukaryotic cells probably use hundreds of GTPases to control different processes, the members of the Ras superfamily have emerged as key players in the regulation of many important biological processes including growth and differentiation, morphogenesis, cell division and motility, cytokinesis, and trafficking through the Golgi, nucleus, and endosomes [2]. Small GTPases are low-molecular-weight (Mr of 20–25 kDa) monomeric guanine nucleotide-binding proteins. They display a conserved structural backbone of 5 G-boxes involved in GTP-binding and GTPase activity [3]. The Ras-related small GTPases are divided into five subfamilies: Ras, Rho, Arf, Rab, and Ran. Ras family members are activated by diverse extracellular stimuli that trigger a series of intracellular signaling events. This cascade of events eventually controls gene transcription, which leads to activation of fundamental cellular processes, including cell growth and differentiation. Ras proteins, the first small GTPases discovered [4], regulate cell growth, proliferation, and differentiation [5,6]. Rho proteins (Rho, Rac, and Cdc42) control the assembly and organization of the actin cytoskeleton, which vastly influences cell morphology [7], while Rab and Arf represent key regulators of secretory and endocytic pathways during vesicle trafficking and microtubule dynamics [8,9]. Ran is the last discovered GTPase and has a central role Cells 2020, 9, 2279; doi:10.3390/cells9102279 www.mdpi.com/journal/cells Cells 2020, 9, x FOR PEER REVIEW 2 of 28 Cells 2020, 9, 2279 2 of 27 during vesicle trafficking and microtubule dynamics [8,9]. Ran is the last discovered GTPase and has a central role in the translocation of RNA and proteins through the nuclear pore complex [10]. inNumerous the translocation studies suggest of RNA a andvery proteins complex through functional the diversity nuclear pore of the complex Ras protein [10]. Numeroussuperfamily. studies Their suggestconserved a very G-domain complex structural functional backbone diversity ofinvolved the Ras proteinin GTP superfamily.-binding and Their GTPase conserved activity G-domain enables structuralcommon biochemical backbone involvedproperties in, while GTP-binding each of them and recognizes GTPase activity its individual enables binding common partners biochemical [6,11]. properties,The diversity while of the each Ras of superfamily them recognizes is demonstrated its individual by bindingthe multiplicity partners of [ 6their,11]. upstream The diversity regulators of the Rasand superfamilydownstream istarget demonstrated proteins to by wh theich multiplicity the Ras-related of their small upstream GTPases regulators bind [12,13 and]. downstream target proteins to which the Ras-related small GTPases bind [12,13]. 1.1. The Rho Family of Proteins—Rho GTPases 1.1. The Rho Family of Proteins—Rho GTPases Rho GTPases are small G proteins found in all eukaryotes [14]. Rho family members are divided Rho GTPases are small G proteins found in all eukaryotes [14]. Rho family members are divided into nine subfamilies (Rho, Rac, Cdc42, RhoDF, Rnd, RhoUV, RhoH, RhoBTB, and Miro) according into nine subfamilies (Rho, Rac, Cdc42, RhoDF, Rnd, RhoUV, RhoH, RhoBTB, and Miro) according to their similar but not identical properties, such as primary amino acid sequence, structural motifs to their similar but not identical properties, such as primary amino acid sequence, structural motifs and biological functions. Similar to other Ras-like GTPases, Rho GTPases are active when bound to and biological functions. Similar to other Ras-like GTPases, Rho GTPases are active when bound to GTP and inactive when bound to GDP. Once activated, they bind effectors that mediate the cellular GTP and inactive when bound to GDP. Once activated, they bind effectors that mediate the cellular response [3]. The Rho signaling module is a complex regulatory network that includes over 240 response [3]. The Rho signaling module is a complex regulatory network that includes over 240 proteins proteins in human [15]. The Rho-family members are defined by the presence of a Rho-specific insert in human [15]. The Rho-family members are defined by the presence of a Rho-specific insert located located between the G4 and the G5 boxes, involved in binding to effectors and regulators [16–18]. between the G4 and the G5 boxes, involved in binding to effectors and regulators [16–18]. Typical Rho Typical Rho proteins are usually low-molecular-weight proteins and consist only of the GTPase proteins are usually low-molecular-weight proteins and consist only of the GTPase domain (conserved domain (conserved structural backbone of 5 G-boxes) and short N- and C-terminal extensions (Figure structural backbone of 5 G-boxes) and short N- and C-terminal extensions (Figure1). 1). FigureFigure 1. SchematicSchematic representation representation of Rho GTPases. GTPases. Colored Colored boxes boxes represent represent characteristic characteristic structures: structures: the Rho GTPase domain (light(light blue),blue), CAAX box (black),(black), BTB (broad(broad complexcomplex//tramtracktramtrack//bbric-a-brac)ric-a-brac) domain I and II (violet),(violet), EFH (EF-hand(EF-hand calcium binding)binding) domain 1 and 2 (green),(green), and the second GTPase domain in Miro (orange). Within theirtheir GTPase GTPase domains, domains, Rho Rho family family members members show show approximately approximately 30% amino 30% acidamino identity acid withidentity other with Ras other proteins Ras proteins and 40–95% and identity40–95% withinidentity the within Rho familythe Rho [7 family]. The human[7]. The genome human encodesgenome 22encodes Rho GTPases 22 Rho GTPases (Table1)[ (Table19]. The1) [19 majority]. The majority of the functional of the functional information information on the on Rho the family Rho family came fromcame studiesfrom studies of typical of typical Rho GTPases: Rho GTPases: RhoA, RhoA, Rac1, Rac1 and, Cdc42 and Cdc42 [7]. All [7] three. All three of them of them regulate regulate actin dynamicsactin dynamics and cytoskeleton and cytoskeleton reorganization reorganization that e ffthatect celleffect shape cell shape and motility and motility [20,21 ].[20 Members,21]. Members of the Rhoof the family Rho family power power the reorganization the reorganization of the actin of the cytoskeleton actin cytoskeleton as a response as a response to external to stimuli,external a stimuli,ffecting aaffecting broad array a broad of cellular array processes of cellular and characteristicsprocesses and such characteristics as adhesion, such migration, as adhesion, proliferation, migration, shape, permeability,proliferation, shape, and polarity permeability [15]. The, and members polarity of [15 the]. The RhoBTB members subfamily of the displayRhoBTB additional subfamilyatypical display features:additional they atypical are muchfeatures: larger they than are much the typical larger GTPases,than the typical and they GTPa haveses, additionaland they have domains additional [22]. Thedomains RhoBTB [22] has. The two RhoBTB so-called has broad two complexso-called/tramtrack broad complex//bric-a-bractramtrack/ (BTB)b domainsric-a-brac found (BTB) to domains mediate homomericfound to mediate or heteromeric homomeric protein–protein or heteromeric interactions protein–protein (Figure 1interactions). The RhoBTB (Figure functions 1). The as aRhoBTB tumor suppressorfunctions as [ 22a tumor]. The