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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 protein-effector 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.

SUBMITTER: Martemyanov KA

PROVIDER: S-EPMC2634932 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Functional comparison of RGS9 splice isoforms in a living cell.

Martemyanov Kirill A KA Krispel Claudia M CM Lishko Polina V PV Burns Marie E ME Arshavsky Vadim Y VY

Proceedings of the National Academy of Sciences of the United States of America 20081219 52

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 ...[more]

PMID: 19098104

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Project description:Alternative splicing allows a single gene to produce multiple transcript-level splice isoforms from which the translated proteins may show differences in their expression and function. Identifying the major functional or canonical isoform is important for understanding gene and protein functions. Identification and characterization of splice isoforms is a stated goal of the HUPO Human Proteome Project and of neXtProt. Multiple efforts have catalogued splice isoforms as "dominant", "principal", or "major" isoforms based on expression or evolutionary traits. In contrast, we recently proposed highest connected isoforms (HCIs) as a new class of canonical isoforms that have the strongest interactions in a functional network and revealed their significantly higher (differential) transcript-level expression compared to nonhighest connected isoforms (NCIs) regardless of tissues/cell lines in the mouse. HCIs and their expression behavior in the human remain unexplored. Here we identified HCIs for 6157 multi-isoform genes using a human isoform network that we constructed by integrating a large compendium of heterogeneous genomic data. We present examples for pairs of transcript isoforms of ABCC3, RBM34, ERBB2, and ANXA7. We found that functional networks of isoforms of the same gene can show large differences. Interestingly, differential expression between HCIs and NCIs was also observed in the human on an independent set of 940 RNA-seq samples across multiple tissues, including heart, kidney, and liver. Using proteomic data from normal human retina and placenta, we showed that HCIs are a promising indicator of expressed protein isoforms exemplified by NUDFB6 and M6PR. Furthermore, we found that a significant percentage (20%, p = 0.0003) of human and mouse HCIs are homologues, suggesting their conservation between species. Our identified HCIs expand the repertoire of canonical isoforms and are expected to facilitate studying main protein products, understanding gene regulation, and possibly evolution. The network is available through our web server as a rich resource for investigating isoform functional relationships (http://guanlab.ccmb.med.umich.edu/hisonet). All MS/MS data were available at ProteomeXchange Web site (http://www.proteomexchange.org) through their identifiers (retina: PXD001242, placenta: PXD000754).

Project description:Two splice isoforms of rabbit alpha(1a)-adrenergic receptor (AR), (named alpha(1a)-OCU.2-AR and alpha(1a)-OCU.3-AR) have been isolated from the liver cDNA library in addition to the previously reported isoform (alpha(1a)-OCU.1-AR). Although they have the identical splice position with human alpha(1a)-AR isoforms, the C-terminal sequences are distinct from those of human isoforms. Among these rabbit alpha(1a)-AR isoforms, there are no significant differences in pharmacological properties: high affinity for prazosin, WB4101, KMD-3213 and YM617 and low affinity for BMY7378, using COS-7 cells expressing each isoform by radioligand binding assay. Competitive reverse transcription-polymerase chain reaction (RT - PCR) analysis revealed that mRNA of alpha(1a)-ARs was expressed in liver, thoracic aorta, brain stem and thalamus of rabbit. The splice isoforms exhibited a distinct distribution pattern in rabbit; alpha(1a)-OCU. 1-AR was expressed most abundantly in those tissues. CHO clones, stably expressing each isoforms with receptor density 740 fmol mg(-1) protein in alpha(1a)-OCU.1-AR, 1200 fmol mg(-1) in alpha(1a)-OCU.2-AR and 570 fmol mg(-1) in alpha(1a)-OCU.3-AR, respectively, showed a noradrenaline-induced increase in inositol trisphosphate which was suppressed by prazosin. Noradrenaline elicited a concentration-dependent increase in extracellular acidification rate (EAR) in the CHO clones with pEC(50) values of 6. 19 for alpha(1a)-OCU.1-AR, 6.49 for alpha(1a)-OCU.2-AR and 6.58 for alpha(1a)-OCU.3-AR, respectively. Noradrenaline caused a concentration-dependent increase in intracellular Ca(2+) concentration ([Ca(2+)]i) in the CHO clones with pEC(50) values of 6. 14 for alpha(1a)-OCU.1-AR, 7.25 for alpha(1a)-OCU.2-AR and 7.70 for alpha(1a)-OCU.3-AR, respectively. In conclusion, the present study shows the occurrence of three splice isoforms of rabbit alpha(1a)-AR, which are unique in C-terminal sequence and in tissue distribution. They show similar pharmacological profiles in binding studies but alpha(1a)-OCU.3-AR had the highest potency of noradrenaline in functional studies in spite of the lowest receptor density. These findings suggest that the structure of C-terminus of alpha(1a)-ARs may give the characteristic functional profile.

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Functional comparison of RGS9 splice isoforms in a living cell.

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Functional comparison of RGS9 splice isoforms in a living cell.

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