Protein Profiling of RGS6, a Pleiotropic Gene Implicated in Numerous Neuropsychiatric Disorders, Reveals Multi-Isoformic Expression and a Novel Brain-Specific Isoform

Abstract

A metanalysis identified Regulator of G protein Signaling 6 (RGS6) as one of 23 loci with pleiotropic effects on four or more human psychiatric disorders. This finding is significant as it confirms/extends the findings of numerous other studies implicating RGS6 in CNS function and pathology. RGS6 is a highly conserved member of the RGS protein family whose cellular roles are likely affected by mRNA splicing and alternative domain inclusion/exclusion. Indeed, we previously identified multiple RGS6 splice variants predicted to produce 36 distinct protein isoforms containing either long (RGS6L) or short (RGS6S) N-terminal domains, an incomplete or intact GGL domain, and 9 alternative C-termini. Unfortunately, sequence similarities between the isoforms have made it difficult to confirm their individual existence and/or to determine their unique functions. Here, we developed 3 RGS6-specific antibodies that recognize all RGS6 protein isoforms (RGS6-fl), the N-terminus of RGS6L isoforms (RGS6-L), and an 18 amino acid alternate C-terminal sequence (RGS6-18). Using these antibodies, we demonstrate that RGS6L(+GGL) isoforms, predominating in both mouse (both sexes) CNS and peripheral tissues, are most highly expressed in the CNS. We further identify 3 novel RGS6 protein bands that are larger (61, 65, and 69kDa) than the ubiquitously expressed 53-57kDa RGS6L(+GGL) proteins. Importantly, we show that the 69kDa protein is a brain-specific dephospho-form of the 65 kDa band, the first identified phosphorylated RGS6 isoform. Together, these data begin to define the functional significance behind the complexity of RGS6 gene processing and further clarifies RGS6's physiological roles by resolving tissue-specific RGS6 protein expression. 3 Significance StatementPsychiatric disorders are highly associated with polygenic variation. Consistent with this, a SNP (rs2332700) in Regulator of G protein Signaling 6 (RGS6) is linked to autism spectrum disorder, bipolar disorder, major depression, and schizophrenia. RGS6 is a highly conserved gene whose complex alternative mRNA splicing produces numerous protein isoforms with high sequence similarity, hindering their functional characterization. Therefore, while aberrant RGS6 signaling and/or expression have been linked to various neuropsychiatric disorders, it is unclear which isoforms are important. This study functionally delineates between the various RGS6 isoforms within mouse. We demonstrate that RGS6 is most highly expressed in CNS, characterize the predominant isoforms, and identify a brain-specific RGS6 protein highly expressed in brain regions associated with various psychiatric disorders.

Regulator of G protein signaling 6 (RGS6) in ventral tegmental area (VTA) dopamine neurons promotes EtOH seeking, behavioral reward and susceptibility to relapse

“…Tissue sections were blocked for 1hr at 4 ° C in 1X PBS + 10% goat serum + 0.5% Triton X-100 (blocking solution). After blocking, tissue sections were incubated overnight at 4 ° C in rabbit-anti-RGS6 (homemade polyclonal antibody against the entire RGS6 protein(26, 30, 32, 60), 1:100, mouse-anti-TH (1:100, Millipore, MAB318), or mouse-anti-DAT (1:100, Millipore MAB669) primary antibody diluted in the blocking solution. Following overnight incubation, tissues were washed 5 times at room temperature in 1X PBS and subsequently incubated for 1hr at 4 ° C in Alexa Fluor 488/568/647-conjugated secondary fluorescent antibodies.…”

“…Samples in loading buffer were boiled (5 min) prior to loading into 10% Mini-PROTEAN® TGX Stain-Free Precast Gels (BioRad, Cat# 4568036). Western blots were probed with a home-made rabbit anti-human RGS6 primary antibody (1:10,000) against the whole protein(26, 60), as well as monoclonal DAT (1:1000, Millipore, MAB669), primary antibody. LI-COR Odyssey goat anti-mouse (Cat# 926-32220 and 926-32210) and goat anti-rabbit (Cat# 926-32211 and 926-68071) were used at a 1:5000 dilution for protein visualization with the Odyssey system.…”

RGS6 mediates exercise-induced recovery of hippocampal neurogenesis, learning, and memory in an Alzheimer’s mouse model

“…1A shows that human DG expresses the same major RGS6 isoforms as seen in mice which are absent in the hippocampus of RGS6 global knockout mice. As we recently described (48), the lower 56kDa band represents RGS6L isoforms and the larger 69kDa band represents a novel brain-specific form of RGS6 (RGS6B) that contains the N-terminal sequence our antibody was generated against. There is a marked loss in RGS6L expression in the DG of AD patients (Figs.…”

“…Tissues were formalin-fixed paraffin-embedded (FFPE) and deparaffinized with a series of xylene and EtOH washes prior to incubation. Sections were incubated overnight at 4 O C with rabbit-anti-RGS6 (homemade polyclonal antibody against the entire RGS6 protein (41,42,48), 1:200. Samples were imaged using an Olympus VS200 Slide Scanner (brightfield).…”

“…After blocking, tissue sections were incubated overnight at 4 O C in rabbit-anti-RGS6 (homemade polyclonal antibody against the entire RGS6 protein (41,42,48), 1:100, mouse-anti-GFP (Invitrogen, MA5-15256, 1:250), mouse-anti-DCX (Santa Cruz Sc-271390, 1:200), or mouseanti-NeuN (Millipore MAB377, 1:200) primary antibody diluted in the blocking solution.…”