Regulator of G protein signaling 14 (RGS14) is a multifunctional signaling protein primarily expressed in mouse pyramidal neurons of hippocampal area CA2 where it regulates synaptic plasticity important for learning and memory. However, very little is known about RGS14 protein expression in the primate brain. Here, we validate the specificity of a new polyclonal RGS14 antibody that recognizes not only full-length RGS14 protein in primate, but also lower molecular weight forms of RGS14 protein matching previously predicted human splice variants. These putative RGS14 variants along with full-length RGS14 are expressed in the primate striatum. By contrast, only full-length RGS14 is expressed in hippocampus, and shorter variants are completely absent in rodent brain. We report that RGS14 protein immunoreactivity is found both pre- and postsynaptically in multiple neuron populations throughout hippocampal area CA1 and CA2, caudate nucleus, putamen, globus pallidus, substantia nigra, and amygdala in adult rhesus monkeys. A similar cellular expression pattern of RGS14 in the monkey striatum and hippocampus was further confirmed in humans. Our electron microscopy data show for the first time that RGS14 immunostaining localizes within nuclei of striatal neurons in monkeys. Taken together, these findings suggest new pre- and postsynaptic regulatory functions of RGS14 and RGS14 variants, specific to the primate brain, and provide evidence for unconventional roles of RGS14 in the nuclei of striatal neurons potentially important for human neurophysiology and disease.

Background: RGS14 binds distinct forms of active and inactive G␣ proteins through its RGS domain and GPR motif. Results: Inactive G␣ i1 -GDP binding of the GPR motif does not preclude RGS action on active G␣ o -GTP. Conclusion: RGS14 simultaneously binds active G␣ o and inactive G␣ i1 while retaining GAP activity. Significance: These findings clarify our understanding of how RGS14 integrates signaling by distinct G protein subunits.

Regulator of G protein signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates G protein and H-Ras/MAPkinase signaling pathways to regulate synaptic plasticity important for hippocampal learning and memory. However, to date, little is known about the subcellular distribution and roles of endogenous RGS14 in a neuronal cell line. Most of what is known about RGS14 cellular behavior is based on studies of tagged, recombinant RGS14 ectopically overexpressed in unnatural host cells. Here, we report for the first time a comprehensive assessment of the subcellular distribution and dynamic localization of endogenous RGS14 in rat B35 neuroblastoma cells. Using confocal imaging and 3D-structured illumination microscopy, we find that endogenous RGS14 localizes to subcellular compartments not previously recognized in studies of recombinant RGS14. RGS14 localization was observed most notably at juxtanuclear membranes encircling the nucleus, at nuclear pore complexes (NPC) on both sides of the nuclear envelope and within intranuclear membrane channels, and within both chromatin-poor and chromatin-rich regions of the nucleus in a cell cycle-dependent manner. In addition, a subset of nuclear RGS14 localized adjacent to active RNA polymerase II. Endogenous RGS14 was absent from the plasma membrane in resting cells; however, the protein could be trafficked to the plasma membrane from juxtanuclear membranes in endosomes derived from ER/Golgi, following constitutive activation of endogenous RGS14 G protein binding partners using AlF4¯. Finally, our findings show that endogenous RGS14 behaves as a cytoplasmic-nuclear shuttling protein confirming what has been shown previously for recombinant RGS14. Taken together, the findings highlight possible cellular roles for RGS14 not previously recognized that are distinct from the regulation of conventional GPCR-G protein signaling, in particular undefined roles for RGS14 in the nucleus.

BackgroundWith the goal of learning to induce regeneration in human beings as a treatment for tissue loss, research is being conducted into the molecular and physiological details of the regeneration process. The tail of Xenopus laevis tadpoles has recently emerged as an important model for these studies; we explored the role of the spinal cord during tadpole tail regeneration.Methods and ResultsUsing ultrafast lasers to ablate cells, and Geometric Morphometrics to quantitatively analyze regenerate morphology, we explored the influence of different cell populations. For at least twenty-four hours after amputation (hpa), laser-induced damage to the dorsal midline affected the morphology of the regenerated tail; damage induced 48 hpa or later did not. Targeting different positions along the anterior-posterior (AP) axis caused different shape changes in the regenerate. Interestingly, damaging two positions affected regenerate morphology in a qualitatively different way than did damaging either position alone. Quantitative comparison of regenerate shapes provided strong evidence against a gradient and for the existence of position-specific morphogenetic information along the entire AP axis.ConclusionsWe infer that there is a conduit of morphology-influencing information that requires a continuous dorsal midline, particularly an undamaged spinal cord. Contrary to expectation, this information is not in a gradient and it is not localized to the regeneration bud. We present a model of morphogenetic information flow from tissue undamaged by amputation and conclude that studies of information coming from far outside the amputation plane and regeneration bud will be critical for understanding regeneration and for translating fundamental understanding into biomedical approaches.

RGS14 contains distinct binding sites for both active (GTP bound) and inactive (GDP bound) forms of Gα proteins. The N‐terminal RGS domain binds active Gαi/o‐GTP, whereas the C‐terminal GPR motif binds inactive Gαi1/3‐GDP. The molecular basis for how RGS14 binds different activation states of Gα to integrate G protein signaling is unknown. Here we explored the effects of G protein binding on the GPR motif and the RGS domain, and examined whether RGS14 can functionally interact with two forms of Gα simultaneously. Using cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive Gαi1‐GDP at the plasma membrane (PM), and that cytosolic RGS14 is recruited to the PM by activated Gαo‐AlF4‐. Bioluminescence resonance energy transfer (BRET) studies showed that RGS14 adopts different conformations in live cells when bound to Gα in different activation states. Hydrogen deuterium exchange mass spectrometry (HDX‐MS) revealed RGS14 is a dynamic protein that undergoes allosteric conformational changes when inactive Gαi1‐GDP binds the GPR motif. Pure RGS14 forms a ternary complex with Gαo‐AlF4‐ and an AlF4‐‐insensitive mutant (G42R) of Gαi1‐GDP as observed by size exclusion chromatography and differential HDX‐MS. Finally, a preformed RGS14:Gαi1‐GDP complex exhibits full capacity to stimulate the GTPase activity of Gαo‐GTP, demonstrating that RGS14 can functionally engage two distinct forms of Gα subunits simultaneously. Based on these findings, we propose a working model for how RGS14 integrates multiple G protein signals.