Cloning of a retinally abundant regulator of G-protein signaling (RGS-r/RGS16): genomic structure and chromosomal localization of the human gene

Snow, Bryan E.; Antonio, Laarni; Suggs, Sid; Siderovski, David P.

doi:10.1016/s0378-1119(97)00593-3

Cited by 34 publications

(21 citation statements)

References 28 publications

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“…RGS16 is expressed predominantly in the retina, pituitary, and liver (22)(23)(24). RGS18 is expressed in hematopoietic tissues and lung (25,26), whereas RGS2 and RGS3 are ubiquitously expressed (20,27).…”

Section: Discussionmentioning

confidence: 99%

Functional Characterization of the G Protein Regulator RGS13

Johnson

Druey

2002

Journal of Biological Chemistry

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The signaling cascades evoked by G protein-coupled receptors are a predominant mechanism of cellular communication. The regulators of G protein signaling (RGS) comprise a family of proteins that attenuate G proteinmediated signal transduction. Here we report the characterization of RGS13, the smallest member of the RGS family, which has been cloned from human lung. RGS13 has been found most abundantly in human tonsil, followed by thymus, lung, lymph node, and spleen. RGS13 is a GTPase-activating protein for G␣ i and G␣ o but not G␣ s . RGS13 binds G␣ q in the presence of aluminum magnesium fluoride, suggesting that it bears GTPase-activating protein activity toward G␣ q . RGS13 blocks MAPK activity induced by G␣ i -or G␣ q -coupled receptors. RGS13 also attenuates GTPase-deficient G␣ q (G␣ q QL) mediated cAMP response element activation but not transcription evoked by constitutively active G␣ 12 or G␣ 13 . Surprisingly, RGS13 inhibits cAMP generation elicited by stimulation of the ␤ 2 -adrenergic receptor. These data suggest that RGS13 may regulate G␣ i -, G␣ q -, and G␣ s -coupled signaling cascades.G protein signaling is an effective mechanism of cellular communication during both physiological and pathological conditions (1-3). Regulators of G protein-signaling (RGS) 1 proteins are a relatively new family of proteins that attenuate G protein-mediated pathways by acting as GTPase-activating proteins (GAPs) for G␣ subunits (4). RGS binding stabilizes a G␣ conformation that favors hydrolysis of GTP to GDP, which hastens the termination of active G␣ (5). The second mechanism of G protein inhibition by RGS proteins is effector antagonism in which the RGS protein binds GTP-bound G␣ q and prevents G␣ q /effector interaction (6).RGS proteins share a common domain, the RGS box. This motif of 120 amino acids is highly conserved in all RGS family members and conveys the ability of RGSs to bind G proteins (7). Although many RGS proteins exhibit GAP activity toward members of the G␣ i (8, 9) and G␣ q (10) families, only one RGS protein has been shown to possess GAP activity specifically toward G␣ s (11). In addition to the classical RGS family, an additional group of proteins exists termed the RhoGEF RGSs (rgRGS) (12, 13). These proteins, which contain a highly diverged RGS homology domain, act as GAPs specifically for G␣ 12/13 and also stimulate GTP binding by Rho family members.Although all RGS proteins contain the conserved RGS box, they display significant variability in sequences outside of this region. For example, a cysteine string found in RGS-GAIP and RGS20 is a site of palmitoylation, which may assist in membrane anchorage (14). The G␣ s GAP RGS-PX1 also contains a Phox domain that may be involved in intracellular trafficking (11).Unique motifs in certain RGSs may modify the G protein GTPase cycle or G protein effector stimulation in additional ways. RGS14, for example, contains a "GoLoco" motif that inhibits guanine nucleotide dissociation on G␣ i (15). Most RGS proteins do not exhibit GAP activity toward G␣ s ...

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Section: Discussionmentioning

confidence: 99%

Functional Characterization of the G Protein Regulator RGS13

Johnson

Druey

2002

Journal of Biological Chemistry

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“…cDNAs for RGS11 and various G protein subunits were cloned from human brain or retinal mRNA, from mouse retinal mRNA, or were obtained as described (7,8); all amplified cDNAs were verified by sequencing. Human RGS7 cDNA was a kind gift of Paul F. Worley (Johns Hopkins University).…”

Section: Methodsmentioning

confidence: 99%

A G protein γ subunit-like domain shared between RGS11 and other RGS proteins specifies binding to G _β5 subunits

Snow

Krumins

Brothers

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

257

266

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Regulators of G protein signaling (RGS) proteins act as GTPase-activating proteins (GAPs) toward the ␣ subunits of heterotrimeric, signal-transducing G proteins. RGS11 contains a G protein ␥ subunit-like (GGL) domain between its Dishevelled͞Egl-10͞Pleckstrin and RGS domains. GGL domains are also found in RGS6, RGS7, RGS9, and the Caenorhabditis elegans protein EGL-10. Coexpression of RGS11 with different G ␤ subunits reveals specific interaction between RGS11 and G ␤5 . The expression of mRNA for RGS11 and G ␤5 in human tissues overlaps. The G ␤5 ͞RGS11 heterodimer acts as a GAP on G ␣o , apparently selectively. RGS proteins that contain GGL domains appear to act as GAPs for G ␣ proteins and form complexes with specific G ␤ subunits, adding to the combinatorial complexity of G protein-mediated signaling pathways.Proteins belonging to the RGS (regulators of G protein signaling) family constitute a newly appreciated group of at least 20 mammalian gene products that act as GTPaseactivating proteins (GAPs) on the ␣ subunits of heterotrimeric, signal-transducing G proteins (1-3). As such, RGS proteins can serve as negative regulators of G proteinmediated signaling pathways by speeding the inactivation of GTP-bound G ␣ subunits. Although several members of the RGS family are relatively simple Ϸ25 kDa proteins that contain little more than a characteristic RGS domain, others include modules that impart additional functions. For example, RGS12 can associate in vitro with certain G protein-coupled receptors by virtue of an alternatively spliced PDZ (PSD-95͞ Dlg͞Z0-1) domain (4), and p115, a guanine nucleotide exchange factor for the low-molecular-weight GTPase rho, contains an RGS domain that imparts sensitivity to regulation by G protein ␣ subunits (5, 6).We describe here a novel G protein ␥ subunit-like domain (GGL; pronounced giggle) that is found in several mammalian RGS proteins (RGS6, RGS7, RGS9, and RGS11) and in EGL-10, an RGS protein of Caenorhabditis elegans. The GGL domains of RGS11 and RGS7 interact preferentially with the G protein ␤ 5 subunit, and the complex of RGS11 and ␤ 5 has GAP activity toward the G protein ␣ o subunit. MATERIALS AND METHODSGeneration of Expression Constructs. cDNAs for RGS11 and various G protein subunits were cloned from human brain or retinal mRNA, from mouse retinal mRNA, or were obtained as described (7,8); all amplified cDNAs were verified by sequencing. Human RGS7 cDNA was a kind gift of Paul F. Worley (Johns Hopkins University). cDNAs encoding G protein subunits were subcloned into the mammalian expression vector pcDNA3.1-Zeo (Invitrogen), and G ␥ and RGS protein cDNAs were subcloned in-frame with an N-terminal tandem hemagglutinin (HA)-epitope tag into a modified pcDNA3.1 vector. Recombinant baculoviruses expressing native or hexahistidine-tagged RGS11 or G ␤5 subunits were generated by using the Bac-To-Bac system by following the manufacturer's protocols (Life Technologies, Gaithersburg, MD).In Vitro Transcription and Translation. Reactions were performed using the T...

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“…Cloning of Human RGS12-Oligonucleotides flanking both the open reading frame (sense primer 5Ј-ATATGGCTCCAAGGGAACAATGAG-ACG-3Ј and antisense primer 5Ј-TACGGGGCCAAGGTGGAGGGATC-AG-3Ј) and 3Ј-UTR of hRGS12 (sense primer 5Ј-ATCCCTCCACCTTG-GCCCCGTAAGC-3Ј and antisense primer 5Ј-CTGCTGGGAGCCTCG-CCTCAGTTTC-3Ј) were designed based on cosmid sequences (9) and used to amplify the hRGS12 cDNA from 0.5 ng of Marathon-Ready TM human brain cDNA (CLONTECH) using the Expand TM long template PCR system (Boehringer Mannheim) as described previously for hRGS16 (15). The resulting PCR products were then cloned and sequenced as described previously (15).…”

Section: Methodsmentioning

confidence: 99%

GTPase Activating Specificity of RGS12 and Binding Specificity of an Alternatively Spliced PDZ (PSD-95/Dlg/ZO-1) Domain

Snow¹,

Hall²,

Krumins³

et al. 1998

Journal of Biological Chemistry

206

172

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Regulator of G-protein signaling (RGS) proteins increase the intrinsic guanosine triphosphatase (GTPase) activity of G-protein ␣ subunits in vitro, but how specific G-protein-coupled receptor systems are targeted for down-regulation by RGS proteins remains uncharacterized. Here, we describe the GTPase specificity of RGS12 and identify four alternatively spliced forms of human RGS12 mRNA. Two RGS12 isoforms of 6.3 and 5.7 kilobases (kb), encoding both an N-terminal PDZ (PSD-95/ Dlg/ZO-1) domain and the RGS domain, are expressed in most tissues, with highest levels observed in testis, ovary, spleen, cerebellum, and caudate nucleus. The 5.7-kb isoform has an alternative 3 end encoding a putative C-terminal PDZ domain docking site. Two smaller isoforms, of 3.1 and 3.7 kb, which lack the PDZ domain and encode the RGS domain with and without the alternative 3 end, respectively, are most abundantly expressed in brain, kidney, thymus, and prostate. In vitro biochemical assays indicate that RGS12 is a GTPaseactivating protein for G i class ␣ subunits. Biochemical and interaction trap experiments suggest that the RGS12 N terminus acts as a classical PDZ domain, binding selectively to C-terminal (A/S)-T-X-(L/V) motifs as found within both the interleukin-8 receptor B (CXCR2) and the alternative 3 exon form of RGS12. The presence of an alternatively spliced PDZ domain within RGS12 suggests a mechanism by which RGS proteins may target specific G-protein-coupled receptor systems for desensitization.The mammalian "regulators of G-protein signaling" (RGS) 1 gene family was first identified by sequence and functional similarity to fungal and nematode genes captured in genetic screens for negative regulators of specific G-protein-coupled receptor (GPCR) signals (1-3). In vitro biochemical analyses soon established that this gene family encodes potent accelerators ("GAPs") of the intrinsic GTP hydrolysis activity of Gprotein ␣ subunits, revealing a molecular mechanism by which RGS proteins drive G-proteins into their inactive GDP-bound form and hence down-regulate GPCR signal transduction in vivo (reviewed in Refs. 4 and 5). However, the mechanisms by which individual RGS proteins desensitize pathways activated by particular GPCRs remain to be elucidated. Tightly regulated transcription has been described for RGS1 (3), RGS2 (6), and RGS3-RGS11 (7), and palmitoylation of the cysteine-rich N terminus of G␣-interacting protein (GAIP) has also been observed (8); however, transcriptional regulation and post-translational modifications of particular RGS family members can each only be expected to afford a gross level of intracellular control over the temporal and spatial expression of G␣-directed GAP activity.We and others have hypothesized that regions outside the RGS fold contribute to regulation of G␣ GAP activity and/or targeting of individual RGS proteins to particular receptor signaling pathways (4, 5, 9, 10). Here, we report the GAP activity of RGS12 and identify a PDZ-like N-terminal sequence within two splice forms. PDZ domains...

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Cloning of a retinally abundant regulator of G-protein signaling (RGS-r/RGS16): genomic structure and chromosomal localization of the human gene

Cited by 34 publications

References 28 publications

Functional Characterization of the G Protein Regulator RGS13

Functional Characterization of the G Protein Regulator RGS13

A G protein γ subunit-like domain shared between RGS11 and other RGS proteins specifies binding to G _β5 subunits

GTPase Activating Specificity of RGS12 and Binding Specificity of an Alternatively Spliced PDZ (PSD-95/Dlg/ZO-1) Domain

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Cloning of a retinally abundant regulator of G-protein signaling (RGS-r/RGS16): genomic structure and chromosomal localization of the human gene

Cited by 34 publications

References 28 publications

Functional Characterization of the G Protein Regulator RGS13

Functional Characterization of the G Protein Regulator RGS13

A G protein γ subunit-like domain shared between RGS11 and other RGS proteins specifies binding to G β5 subunits

GTPase Activating Specificity of RGS12 and Binding Specificity of an Alternatively Spliced PDZ (PSD-95/Dlg/ZO-1) Domain

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A G protein γ subunit-like domain shared between RGS11 and other RGS proteins specifies binding to G _β5 subunits