Molecular Cloning of a Novel 69 kDa Brain‐Specific Isoform of Regulator of G Protein Signaling 6 (RGS6)
Authors: Ahlers Katelin E. , Stewart Adele , Yang Jianqi , Koland John G. , Fisher Rory A. 
Published: November 2023

RGS proteins play critical roles in modulating the magnitude and duration of signaling through many G protein‐coupled receptors (GPCRs) due to their ability to facilitate inactivation of heterotrimeric G protein signaling. RGS proteins terminate both Gα and Gβγ signaling through their GTPase‐activating activity towards specific Gα subunits, an activity bestowed by their semiconserved RGS domain. RGS6 is a member of the R7 subfamily of RGS proteins distinguished by two additional domains, DEP/DHEX and GGL, which target R7 members to the membrane and regulate protein stability, respectively. We have demonstrated that RGS6‐specific inhibition of Gαi/o is critical for the modulation of several CNS disorders for which we suggest RGS6 is a novel therapeutic target. Remarkably, we have discovered that RGS6−/− mice have reduced behavioral correlates of anxiety and depression, exhibit diminished alcohol seeking and reward behaviors, and develop late age‐onset Parkinson's disease. The role of RGS6 in these disorders is dependent on its ability to inhibit the signaling of various brain GPCRs, including: 5‐HT1A serotonin receptors in hippocampal and cortical neurons (anxiety/depression), GABAB receptors in mesolimbic dopaminergic neurons (alcoholism), and D2 receptors in dopaminergic neurons of the substantia nigra pars compacta (Parkinson's disease). Potentially key to the ability of RGS6 to regulate these various GPCRs are previously unidentified domains that arise via alternative mRNA splicing. Our initial cloning efforts identified 36 distinct splice‐forms in human brain encoding RGS6 isoforms ≤ 56 kDa. Recently, we identified, in both mouse and humans, at least two additional uniquely brain‐specific RGS6 protein isoforms that are larger (~61 and 69 kDa) than the ubiquitously expressed 56 kDa forms. The functions of these RGS6 variants and how they arise is unknown, but they may be critical for our understanding CNS pathology as both isoforms are highly expressed in the brain regions affected by the CNS disorders referred to above. Here we report PCR amplification and cloning of six novel RGS6 cDNAs from a human brain library that arise by alternative mRNA splicing and inclusion of novel RGS6 exons. One of these novel cDNAs encodes a 69 kDa protein that co‐migrates with the large brain‐specific RGS6 protein present in human and mouse. This RGS6 splice‐form exhibits a near exclusive CNS pattern of expression and generates an RGS6 protein with a predicted C‐terminal extension near the RGS domain that may represent a novel protein interaction site. Other identified RGS6 cDNAs include those with novel exons that lead to early stop codons yielding RGS6 proteins lacking all but the N‐terminal domain and those with novel 3′ exons that generate RGS6 splice‐forms with unique C‐terminal domains. Interestingly, several of these newly identified exons are shared primarily between humans and other primates. Together, this research lays the foundation for experiments to elucidate the functional significance of RGS6 alternative mRNA splicing in normal brain function as well as pathology.Support or Funding InformationNIH CA161882, MJ Fox 11551