RGS12 Interacts with the SNARE-binding Region of the Cav2.2 Calcium Channel

Abstract

Activation of GABA B receptors in chick dorsal root ganglion (DRG) neurons inhibits the Ca v 2.2 calcium channel in both a voltage-dependent and voltage-independent manner. The voltage-independent inhibition requires activation of a tyrosine kinase that phosphorylates the ␣ 1 subunit of the channel and thereby recruits RGS12, a member of the "regulator of G protein signaling" (RGS) proteins. Here we report that RGS12 binds to the SNARE-binding or "synprint" region (amino acids 726 -985) in loop II-III of the calcium channel ␣1 subunit. A recombinant protein encompassing the Nterminal PTB domain of RGS12 binds to the synprint region in protein overlay and surface plasmon resonance binding assays; this interaction is dependent on tyrosine phosphorylation and yet is within a sequence that differs from the canonical NPXY motif targeted by other PTB domains. In electrophysiological experiments, microinjection of DRG neurons with synprint-derived peptides containing the tyrosine residue Tyr-804 altered the rate of desensitization of neurotransmitter-mediated inhibition of the Ca v 2.2 calcium channel, whereas peptides centered about a second tyrosine residue, Tyr-815, were without effect. RGS12 from a DRG neuron lysate was precipitated using synprint peptides containing phosphorylated Tyr-804. The high degree of conservation of Tyr-804 in the SNAREbinding region of Ca v 2.1 and Ca v 2.2 calcium channels suggests that this region, in addition to the binding of SNARE proteins, is also important for determining the time course of the modulation of calcium current via tyrosine phosphorylation.Multiple G protein-mediated signaling pathways are known to modulate Ca v 2.2 (N-type) calcium channels (1, 2) via direct G protein-ion channel interactions, activation of second messenger cascades, and activation of tyrosine kinases (3, 4). This modulation of voltage-dependent calcium channels is a transient phenomenon. Upon prolonged exposure to a neurotransmitter, neurons become unresponsive or desensitized. Despite the common requirement for the activation of a G proteincoupled receptor kinase (GRK3) for desensitization of the neurotransmitter-mediated inhibition of calcium current (5), G i -and G o -mediated pathways exhibit different rates of desensitization (6) that may result from selective effects of the G␣-directed GTPase-accelerating activity borne by "regulator of G protein signaling" (RGS) 1 proteins (7,8).In dorsal root ganglion (DRG) neurons, the activation of ␥-aminobutyric acid type B (GABA B ) receptors induces both voltage-dependent and voltage-independent inhibition of Ca v 2.2 channels (9). Voltage-independent inhibition requires the activation of a tyrosine kinase that phosphorylates the pore-forming ␣-subunit of the calcium channel (10). The tyrosine-phosphorylated form of the ␣-subunit becomes a target for the phosphotyrosine binding (PTB) domain of RGS12, a member of the RGS protein superfamily that specifically accelerates the rate of desensitization of this response (10).To better understand the molecular b...

Differential expression of regulator of G‐protein signaling R12 subfamily members during mouse development

“…Consistent with this evidence, the long form of RGS12, through its PTB domain, is capable of direct, neurotransmitter-dependent interactions with the tyrosine-phosphorylated N-type calcium channel in primary dorsal root ganglion neurons (Schiff et al, 2000;Richman et al, 2005). The differential expression patterns of RGS12 splice variants in the embryo support our hypothesis that these proteins may serve a significant role in signal modification and scaffolding, presumably allowing specific subsets of signaling proteins to interact in specific locations during development.…”

“…In addition to the RGS/RBD/GoLoco domain core, the long RGS12 splice variant has an N-terminal PDZ (PSD-95/Dlg/ZO-1) domain capable of binding the interleukin (IL) -8 receptor B (CXCR2) or its own C-terminus (Snow et al, 1998) and a phosphotyrosinebinding (PTB) domain that associates with tyrosine-phosphorylated N-type calcium channels (Schiff et al, 2000;Richman et al, 2005). Therefore, RGS12, in at least one isoform, may also mediate tyrosine kinase-dependent regulation of calcium channels and coordinate this regulation with Gprotein signals.…”

Upregulation of PSCDBP, TLR2, TWIST1, FLJ35382, EDNRB, and RGS12 Gene Expression in Human Myometrium at Labor

“…430 RGS12 interacts with both N-type and Ca(v)2.2 Ca 2+ channels and also binds a C-terminal motif found in proteins such as the IL-8 receptor. [39][40][41] We observed a  5-fold change in RGS12 mRNA expression between non-laboring and laboring myometrium. Western blotting demonstrated expression of RGS12 in human myometrial smooth muscle cells and immunofluorescence microscopy localized it to the cell cytoplasm.…”

G protein inhibition of CaV2 calcium channels

“…However, RGS proteins have several other modes of action 172 including some that dimerize with Gβ 5 subunits and some (e.g., RGS12) that directly interact with Ca V 2.2 channels. 86,173 Experimental manipulation of RGS expression has been demonstrated to alter calcium channel inhibition 174,175 and it has also been shown that this in turn can alter short term synaptic plasticity. 176 Expression of several RGS proteins can be dynamically regulated by physiological and pathophysiological situations.…”