The RGK family of GTP-binding proteins: regulators of voltage-dependent calcium channels and cytoskeleton remodeling

Abstract

RGK proteins constitute a novel subfamily of small Ras-related proteins that function as potent inhibitors of voltage-dependent (VDCC) Ca(2+) channels and regulators of actin cytoskeletal dynamics. Within the larger Ras superfamily, RGK proteins have distinct regulatory and structural characteristics, including nonconservative amino acid substitutions within regions known to participate in nucleotide binding and hydrolysis and a C-terminal extension that contains conserved regulatory sites which control both subcellular localization and function. RGK GTPases interact with the VDCC beta-subunit (Ca(V)beta) and inhibit Rho/Rho kinase signaling to regulate VDCC activity and the cytoskeleton respectively. Binding of both calmodulin and 14-3-3 to RGK proteins, and regulation by phosphorylation controls cellular trafficking and the downstream signaling of RGK proteins, suggesting that a complex interplay between interacting protein factors and trafficking contribute to their regulation.

Figure 1. RGK proteins interact with a variety of factors to regulate their localization and functions

Human RGK proteins were aligned using ClustalW and columns are scored as “*” (residues identical among aligned sequences), “:” (conserved substitutions among aligned sequences), or “.” (semi-conserved substitutions among aligned sequences). The positions of Ca_Vβ accessory channel subunits (through the GTPase core), calmodulin (CaM), and PIP lipid binding are indicated. Residues highlighted in green are known to reduce or eliminate CaM binding, while those highlighted in teal (basic residues) and orange (hydrophobic residues) are proposed to be involved in PIP lipid binding. Residues highlighted in red are phosphorylation sites that have been directly confirmed through experimentation with the exception of S18/S290 in mouse Rem (and corresponding residues in other RGK proteins) that are thought to be phosphorylated based on their roles in 14-3-3 association.

Figure 2. Proposed mechanisms for Gem/Rad mediated cytoskeleton reorganization

Gem/Rad has been proposed to antagonize Rho GTPase signaling at two levels. First, Gem has been shown to interact with ROKβ, which inhibits (grey arrows) the phosphorylation of myosin light chain (MLC) and myosin binding subunit (MBS) of the myosin phosphatases, but not (black arrow) LIM kinase. Rad interacts with ROKα and induces similar cellular effects, likely through a similar mechanism. Moreover, GTP-bound Gem has also been shown to interact with Ezrin at the plasma membrane-cytoskeleton interface and recruit the Gmip RhoGAP to down-regulate RhoA signaling.

Figure 3. Potential mechanisms used by RGK proteins inhibit the function of voltage-dependent Ca²⁺ channels

A) Chronic regulation of VDCC function by altered trafficking. Left, newly-synthesized Ca_Vαsubunits are retained in the endoplasmic reticulum (ER) until association with a Ca_Vβ subunit promotes trafficking to the plasma membrane. Center, Ca_Vβ subunits have been found to simultaneously associate with both Ca_Vαand RGK proteins. This scaffolded complex may serve to inhibit channel trafficking. Right, RGK proteins have also been demonstrated to bind and sequester Ca_Vβ subunits. In this model, the RGK/Ca_Vβ complex never associates with newly-synthesized Ca_Vα subunits which remain trapped in the ER. B) Acute regulation of VDCC at the plasma membrane by RGK proteins. Acute regulation is proposed to involve two independent molecular events, association of RGK proteins with Ca_Vβ/Ca_Vα, and membrane anchoring of the conserved RGK C-terminus. Membrane association of the C-terminus involves the interaction of the positively charged RGK polybasic region with negatively charged inositol 1,4,5-bisphosphate and –trisphosphate lipids (PIP lipids).