RGS6 Protein Profiling Identifies Multi‐isoform Expression in Mouse Tissues and a Novel Brain‐specific Isoform
Authors: Ahlers‐Dannen Katelin , Yang Jianqi , Spicer Mackenzie , Maity Biswanath , Stewart Adele , Fisher Rory 
Published: June 2021

RGS proteins modulate the magnitude and duration of G protein‐coupled receptor (GPCR) signaling by facilitating heterotrimeric G protein inactivation, a function bestowed by their RGS domain. RGS6 is a member of the R7 subfamily distinguished by two additional domains, DEP and GGL, which target these proteins to the membrane and promote their stability, respectively. In addition to DEP‐ and GGL‐mediated spatial and functional regulation, RGS6's cellular roles are also likely affected by mRNA splicing and alternative domain inclusion/exclusion. Indeed, we previously identified multiple RGS6 splice variants predicted to produce 36 distinct RGS6 protein isoforms containing either long (RGS6L) or short (RGS6S) N‐terminal domains, an incomplete or intact GGL domain, and 9 alternative C‐terminal sequences. While sequence similarities have complicated the study of individual RGS6 protein isoforms, we and others have demonstrated that RGS6‐specific inhibition of GPCR‐Gαi/o signaling is critical for the modulation of several CNS disorders and cardiac function/dysfunction. Furthermore, RGS6 has unique G protein‐independent functions that are required for its beneficial tumor suppressor role as well as its detrimental roles in mediating alcohol‐induced peripheral toxicity. As the list of RGS6's critical physiological and pathophysiological functions continues to grow, it seemed imperative to conduct a comprehensive analysis of mouse whole‐body RGS6 protein isoform expression. Therefore, we developed RGS6‐specific antibodies that recognize all RGS6 protein isoforms (RGS6‐fl), that selectively detect the N‐terminus of RGS6L isoforms (RGS6‐L), and that detect an 18 amino acid alternate C‐terminal sequence (RGS6‐s) present in 56% of predicted RGS6 proteins. Using these antibodies we demonstrated that RGS6 proteins are most highly expressed in CNS tissues, but are also expressed in: lung, kidney, bladder, prostate, heart, omental fat, stomach, intestine, and breast. Furthermore, western analysis revealed that, while RGS6 proteins with MWs corresponding to RGS6L+GGL isoforms were expressed in multiple tissues, RGS6L‐GGL and RGS6S isoforms were not detected, suggesting they are less stable or are expressed at much lower levels than RGS6L+GGL isoforms. Finally, western analysis identified two novel brain‐specific RGS6 protein isoforms of unknown origin and function that are larger (~61 and 69kDa) than the ubiquitously expressed ~56kDa RGS6L proteins. Both RGS6 isoforms are recognized by the RGS6‐L and RGS6‐fl antibodies while only the 69 kDa isoform is detected by RGS6‐s. Interestingly, the 69kDa isoform appears to a brain‐specific RGS6 isoform. Together, these data begin to define the functional significance behind the complexity of RGS6 gene processing and further clarifies RGS6's role in normal physiology and pathophysiology by resolving tissue‐specific RGS6 protein expression.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.