Abstract
RGK proteins, Gem, Rad, Rem1, and Rem2, are members of the Ras superfamily of small GTP-binding proteins that interact with Ca2+ channel β subunits to modify voltage-gated Ca2+ channel function. In addition, RGK proteins affect several cellular processes such as cytoskeletal rearrangement, neuronal dendritic complexity, and synapse formation. To probe the phylogenetic origins of RGK protein–Ca2+ channel interactions, we identified potential RGK-like protein homologs in genomes for genetically diverse organisms from both the deuterostome and protostome animal superphyla. RGK-like protein homologs cloned from Danio rerio (zebrafish) and Drosophila melanogaster (fruit flies) expressed in mammalian sympathetic neurons decreased Ca2+ current density as reported for expression of mammalian RGK proteins. Sequence alignments from evolutionarily diverse organisms spanning the protostome/deuterostome divide revealed conservation of residues within the RGK G-domain involved in RGK protein – Cavβ subunit interaction. In addition, the C-terminal eleven residues were highly conserved and constituted a signature sequence unique to RGK proteins but of unknown function. Taken together, these data suggest that RGK proteins, and the ability to modify Ca2+ channel function, arose from an ancestor predating the protostomes split from deuterostomes approximately 550 million years ago.
Highlights
The RGK protein family, comprised of Gem, Rad, Rem1, and Rem2, is an atypical subset of the Ras superfamily [1] of small GTP-binding proteins
We provide evidence for RGK-like protein homologs in the genomes of non-vertebrate deuterostomes and protostomes suggesting that a common RGK protein ancestor arose prior to the split between deuterostomes and protostomes approximately 550 million years ago
Expression of cloned RGK protein ortholog/homolog open reading frames from Danio rerio and Drosophila melanogaster mRNA in mammalian neurons decreased ICa density establishing conservation of this phenotype
Summary
The RGK protein family, comprised of Gem, Rad, Rem, and Rem, is an atypical subset of the Ras superfamily [1] of small GTP-binding proteins. Rad was initially cloned based on differential mRNA expression in type II diabetic muscle [2]. The connection between RGK proteins and voltage-gated Ca2+ channel (VGCC) function arose from a yeast-two hybrid screen for protein partners that interact with VGCC b-subunits (Cavb). Subsequent studies confirmed and extended these observations demonstrating that all four RGK family members interacted with all four Cavb subunits (CACNB1-4) to attenuate VGCC function [8,9,10,11]. Additional mechanisms underlying attenuation of VGCC have been proposed including production of a non-conducting species [12,13,14], disruption of channel gating [15], and binding to the a1-subunit of some VGCCs [16]. Genetic ablation of Rem [17] or Rad [18] in mice results in increased L-type Ca2+ current in cardiac myocytes suggesting that endogenous RGK proteins tonically inhibit VGCC function in this tissue
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