Functional comparison of RGS9 splice isoforms in a living cell

Abstract

Two isoforms of the GTPase-activating protein, regulator of G protein signaling 9 (RGS9), control such fundamental functions as vision and behavior. RGS9 -1 regulates phototransduction in rods and cones, and RGS9 -2 regulates dopamine and opioid signaling in the basal ganglia. To determine their functional differences in the same intact cell, we replaced RGS9 -1 with RGS9 -2 in mouse rods. Surprisingly, RGS9 -2 not only supported normal photoresponse recovery under moderate light conditions but also outperformed RGS9 -1 in bright light. This versatility of RGS9 -2 results from its ability to inactivate the G protein, transducin, regardless of its effector interactions, whereas RGS9 -1 prefers the G proteineffector complex. Such versatility makes RGS9 -2 an isoform advantageous for timely signal inactivation across a wide range of stimulus strengths and may explain its predominant representation throughout the nervous system. G proteins ͉ phototransduction ͉ RGS proteins P roteins of the regulators of G protein signaling (RGS) family are ubiquitous regulators of signal duration in many G protein pathways. Their RGS homology domain is directly responsible for accelerating the GTPase activity of G protein ␣-subunits, but most RGS proteins also contain additional domains, which vary greatly among the family members. Little is known about the functional roles of these non-catalytic domains, although they are thought to contribute to the specificity of RGS interactions (reviewed in 1, 2). RGS9 is one of the better-studied multidomain RGS proteins and exists in two splice isoforms (3-5), both of which form constitutive complexes with the type 5 G protein ␤ subunit, G␤5 (6-8). The difference between these isoforms resides in the structure of their C-termini: a short 18-aa sequence in RGS9-1 is replaced with 209 residues in RGS9-2 (Fig. 1A).RGS9-1 is expressed exclusively in rod and cone photoreceptors where it sets the duration of electrical responses to light by accelerating the GTPase activity of transducin (3). In mice, the lack of RGS9 causes a drastic delay in photoresponse recovery (9), and its mutation in humans leads to difficulties in adjusting to bright light and seeing moving objects (10). RGS9-2 is expressed predominantly in the striatum, where it controls reward behavior and movement coordination by regulating D2 dopamine and -opioid receptor signaling (11-15). RGS9 knockout mice display augmented sensitivity to rewarding properties of morphine and cocaine (12, 13) and rapidly develop dyskinesias following administration of dopamine receptor antagonists (15).The physiological significance of having two RGS9 isoforms is unknown. High-affinity interaction of RGS9-1 with transducin requires that transducin first binds its effector, the ␥-subunit of cGMP phosphodiesterase (PDE␥) (16). As a result, both RGS9-1 (9) and PDE␥ (17) are needed for timely GTP hydrolysis by transducin and normal recovery of the rod from light excitation. We hypothesized that the biological role for such a dual requirement is to ensure ...

Membrane Attachment Is Key to Protecting Transducin GTPase-Activating Complex from Intracellular Proteolysis in Photoreceptors

“…5A,C), with levels ranging between 1.7 and 4.9-fold above the wild type RGS9-1 content. We noted, however, that measurements of protein content had unusual variability among mice of the same line, leading us to suggest that rods from ggR9AP mice exhibit mosaicism in their expression of the transgene, as occasionally observed in transgenic lines using this promoter (Lem et al, 1991) and encountered in one of our recent studies (Martemyanov et al, 2008). Staining of R9AP −/− ggR9AP retinal flatmounts for R9AP revealed that this was indeed the case, as patches of rhodopsin-positive outer segments without R9AP expression were easily identified (Fig.…”

Implications of dimeric activation of PDE6 for rod phototransduction

“…The second, longer ‘dominant time constant’ (of 600–800 ms) observed in electrophysiological measurements on mouse rods has been proposed to reflect the time constant of such decay of free G* in vivo , on the basis of experiments incorporating RGS9-2 into rods [42]. We concur with that interpretation, and note that it conforms with the biochemical finding that, in the presence of the RGS domain of RGS9 but in the absence of PDE γ , the rate of GTP hydrolysis by activated transducin is around 1 s −1 [43] (in contrast to the much slower rate for purified transducin α-subunits, of around 0.05 s −1 [44]).…”

Chapter 6 Structure, Function, and Localization of Gβ5–RGS Complexes

“…45,47,48 A recent report showed that RGS9-2 can functionally substitute for RGS9-1 in photoreceptors and, in fact, appears to be a more efficient inhibitor of transducin than RGS9-1. 49 …”