GTPases, along with their associated regulators and effectors, play central roles in signal transduction pathways that influence nearly every aspect of cell biology. A significant portion of these proteins belongs to a superfamily named after the RAS oncoprotein. Research into the biochemistry and functions of RAS-related GTPases has concentrated on a relatively small subset of these proteins. Genome analysis and gene expression data from multiple sources have been used to create a comprehensive overview of the genes and proteins that constitute the human RAS superfamily, as well as some more distantly related GTPases. Sequence comparison analysis has provided insights into the relationships among members of this signal transduction superfamily.

Biochemical Characteristics of RAS Superfamily Proteins

RAS superfamily proteins share a fundamental biochemical activity: GTP (guanosine triphosphate) binding and hydrolysis. This commonality is reflected in each protein's several characteristic "G box" sequences. The G1 box, also known as the P-loop or Walker A motif, is a purine nucleotide binding signature. The G3 box, which overlaps with the Walker B motif at the invariant aspartic acid residue, is involved in binding a nucleotide-associated Mg²⁺ ion and is highly conserved among superfamily members. The G4 box residues make hydrogen bond contact with the guanine ring, conferring specificity to GTP over ATP, and provide stabilizing interactions with G1 box residues. The G5 box primarily makes indirect associations with the guanine nucleotide and is less well-conserved among superfamily members. The G2 box, present in several RAS subfamily members, is involved in forming major components of the effector binding surface.

Mechanism of Action

RAS proteins operate through nucleotide-regulated conformational shifts. In the GTP-bound state, they exhibit a high affinity for downstream effector proteins. Structural changes primarily occur in two loop regions called switch 1 and switch 2. The high-affinity effector-binding conformation of RAS proteins is transient; GTP hydrolysis and release of the γ-phosphate lead to reorientation of effector binding residues, release of effector proteins, and attenuation of downstream signaling.

Figure 1. RAS signaling impacts various cellular functions, prominently through the MAPK Raf-MEK-ERK pathway. (Piazza GA, et al., 2024)

In the rapidly evolving field of biotechnology, research into the RAS gene family and associated signaling pathways plays a crucial role in understanding cancer, cell development, and regulation. Investigating these intricate mechanisms often requires advanced tools such as gene editing, viral vectors, and cell line development for precise functional studies. Creative Biogene offers comprehensive research support, including high-quality gene cloning, viral packaging, and stable cell line development.

Regulation of RAS Proteins

The rate-limiting step in RAS protein activation is the exchange of bound GDP for GTP. This step is typically slow, favoring an inactive steady-state conformation of RAS even in the presence of a high cellular GTP/GDP ratio. Guanine nucleotide exchange factors (GEFs) catalyze the release of GDP, promoting GTP loading and activation of RAS. Several GEFs may act on a particular RAS protein, each responding to distinct upstream stimuli, providing multiple avenues for signal regulation. GEF-mediated regulation is a point of vulnerability for RAS function, as RAS mutants binding GEFs unproductively can block the activation of endogenous RAS.

Guanine nucleotide dissociation inhibitors (GDIs) counteract exchange factors by binding specifically to GDP-bound GTPases and inhibiting GDP release, thus prolonging the inactive state. GDIs also emulsify some lipid-modified GTPases, allowing them to dissociate from membrane surfaces. While multiple GDIs have been identified for RHO and RAB proteins, none have been identified for other subfamilies.

The intrinsic GTPase activity of RAS-related proteins is typically low, prolonging signal transduction. GTP hydrolysis is significantly enhanced by GTPase activating proteins (GAPs). Like GEFs, multiple GAPs may function on a given RAS protein, allowing for various input sources at this stage of regulation.

Posttranslational Modifications

Many RAS family proteins undergo multiple lipid modifications, promoting association with cellular membranes. Covalent posttranslational modification of C-terminal cysteine residues by isoprenylation (attachment of a farnesyl or geranylgeranyl group) is observed in most RAS, RHO, and RAB family members. This modification influences subcellular membrane localization, which in turn affects effector binding or activation and regulatory protein interactions. Cysteine palmitoylation also occurs near the C terminus of some RAS and RHO proteins.

At the N terminus of many ARF (ADP ribosylation factor) and Gα subfamily proteins, myristoylation occurs. For some Gα proteins, myristoylation is combined with palmitoylation of a neighboring cysteine. N-terminal lipid additions likely contribute to membrane localization and may impact RAS protein structure or function.

Discovery and Functional Roles

RAS genes were first identified as transduced oncogenes in Harvey and Kirsten strains of acutely transforming retroviruses. Mutationally activated forms of HRAS (H-Ras), KRAS (K-Ras), and NRAS (N-Ras) were later isolated from human tumor cells using transfection-based assays. Tumor-derived RAS mutations, such as HRASG12V, disable GTPase function and GAP responsiveness. Mutations enhancing guanine nucleotide exchange (e.g., HRASN116H) also increase the basal activation state of RAS proteins.

KRAS-activating missense mutations are commonly found in non-small cell lung cancer, colon adenomas, and pancreatic adenocarcinomas, making KRAS the most common mutationally activated human oncoprotein. HRAS- or NRAS-activating mutations are also seen in some tumors. Beyond mutational activation, RAS genes can be amplified or overexpressed in certain tumors. For instance, breast cancer shows low RAS-activating mutation rates but elevated RAS activity due to increased upstream signaling from the receptor tyrosine kinase ERBB2 (Her2). Other mechanisms leading to RAS overactivation include the deletion of genes encoding negative regulators and the overexpression of positive regulators.

Figure 2. Mutant RAS proteins exhibit altered biochemical features. (Moore AR, et al., 2020)

Less Common RAS Superfamily Proteins

Activating mutations in other RAS superfamily members are rare in human tumors. However, in vitro systems provide evidence that several members, beyond KRAS, HRAS, and NRAS, can enhance or facilitate cell transformation.

Subfamily Characteristics

RAS subfamily members show high conservation within the G1, G3, G4, and G5 boxes. Most proteins in this group are relatively small and do not exhibit prominent functional motifs. Most RAS subfamily proteins localize predominantly to the plasma membrane, partly due to C-terminal prenylation. Some RAS subfamily members also contain fatty acid acylation signals. Notably, HRAS, NRAS, ERAS, RRAS1, RAP2A, RAP2B, and RAP2C have palmitoylated cysteine residues proximal to their C-terminal prenylated cysteines. The functional implications of these modifications are still under investigation.

Splicing Variations and Evolution

Alternative splicing of some RAS genes, such as KRAS and HRAS, may generate isoforms with distinct subcellular localizations. Comparisons of RAS subfamily sequences across species show strong evolutionary conservation and suggest an expansion of RAS subfamily proteins, with multiple structural or functional branches.

Key RAS Effectors

HRAS, KRAS, and NRAS proteins are well-known for their mitogenic properties and their role in cell transformation. These proteins also contribute to cell differentiation, organ development, and neuronal plasticity. The protein kinase RAF1 (c-Raf), along with ARAF and BRAF, was the first identified RAS effector. Activated RAS binds with high affinity to Raf-like Ras-binding domains, leading to RAF activation and initiation of the MEK-ERK MAPK cascade, which affects transcription and other cellular functions.

Phosphatidylinositol 4,5 bisphosphate 3-kinase (PI3K) catalytic subunits also represent established RAS effectors. RAS binding to PI3K promotes its catalytic activity, resulting in increased PIP3 production and recruitment of PIP3-binding PH domain proteins such as AKT1 and PDPK1. RAS-mediated activation of PI3K is an important component of cell transformation.

Several GEFs for RAL proteins are RAS effectors, including RALGDS, RGL1, RGL2, and RGL3, which encode Ras association domains. RAS proteins stimulate the nucleotide-exchange activity of RALGDS, contributing to human cell transformation. RIN1 is another RA domain-containing RAS effector protein, functioning as a RAS-responsive GEF for RAB5 and stimulating the catalytic activity of the ABL tyrosine kinase.

The RAS gene family, with its various proteins and effectors, plays a crucial role in cell biology and tumorigenesis. Ongoing research continues to uncover the complexities of RAS functions, their regulation, and their implications in cancer and other diseases.