Specific Interaction of Gαi3 with the Oa1 G-Protein Coupled Receptor Controls the Size and Density of Melanosomes in Retinal Pigment Epithelium

Young, Alejandra; Jiang, Meisheng; Wang, Ying; Ahmedli, Novruz B.; Ramirez, Jessica; Reese, Benjamin E.; Birnbaumer, Lutz; Farber, Débora B.

doi:10.1371/journal.pone.0024376

Cited by 20 publications

(20 citation statements)

References 32 publications

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“…Moreover, recently, it has has been shown to be activated by a non-GPCR dependent mechanism at the Golgi (Lo et al, 2015). Gαi3 is also activated on other subcellular membranes, like LC3-positive autophagosomes (Garcia-Marcos et al, 2011), melanosomes (Young et al, 2011) and at the plasma membrane (Thompson et al, 2007;Kuwano et al, 2016). Thus, the same signaling molecules can operate in different ways in different regulatory networks for different purposes.…”

Section: Discussionmentioning

confidence: 99%

Autoregulatory circuit regulating basolateral cargo export from the TGN: role of the orphan receptor GPRC5A in PKD signaling and cell polarity

Martino

Capalbo

Sticco

et al. 2020

Preprint

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The membrane transport apparatus comprises a series of separate membrane bound compartments, or transport stations, that are responsible for the synthesis, processing, transport, sorting and delivery to their final cellular destinations of most transmembrane and soluble lumenal proteins. Over the last decades the membrane transport system has been shown to be extensively regulated both by environmental inputs and by internal homeostatic signalling systems, or control systems, that operate to maintain the homeostasis and optimal functionality of the main transport stations, such as the endoplasmic reticulum and the Golgi, in the face of internal and external perturbations. The trans-Golgi network (TGN) is a major transport and processing station and the main sorting compartment of the transport apparatus. However, the mechanisms that control cargo export and sorting at the TGN have so far remained elusive. Here we focus on the sorting of basolateral cargo proteins and show that these proteins bind to the TGN localized orphan receptor GPRC5A. The cargo-GPRC5A complex triggers the activation of a signaling pathway that involves the Gβγ subunits dependent activation of the phospholipase C beta 3 (PLCβ3), which inturn induces diacyl glycerol (DAG) production. DAG recruits and activates protein kinase D (PKD) and the phosphorylation of its substrates. This step results in the formation of basolateral carriers for delivery of these cargoes to the basolateral plasma membrane domain. We term this mechanism "ARTG" (AutoRegulation of TGN export). Remarkably, the impairment of ARTG pathway components, and in particular of GPRC5A, causes defects in the polarized organization of epithelial cells.

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Section: Discussionmentioning

confidence: 99%

Autoregulatory circuit regulating basolateral cargo export from the TGN: role of the orphan receptor GPRC5A in PKD signaling and cell polarity

Martino

Capalbo

Sticco

et al. 2020

Preprint

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“…While the precise function of Gαi3 in RPE melanosome biogenesis remains to be delineated, our studies suggest that this protein plays an important role in the control of the size and density of RPE melanosomes and therefore in the regulation of RPE pigmentation [10], [20]. We hypothesize that a constitutively active Gαi3 protein could by-pass the lack of Oa1 in Oa1−/− mice and keep the Oa1 signaling cascade going leading to the normalization of their RPE pigmentation.…”

Section: Introductionmentioning

confidence: 82%

“…We have previously demonstrated by in-vitro and in-vivo studies on mice that Gαi3 physically interacts with the Oa1 protein in the RPE and that lack of Gαi3 from the mouse genome leads to the presence of RPE macromelanosomes and a reduced melanosomal density, suggesting that Gαi3 is the first downstream component in the Oa1 signaling pathway [10], [20]. This work was designed to test the hypothesis that Gαi3 is the major transducer for Oa1-mediated melanogenesis in RPE cells.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, Gαi3 (Q204L) ensures the proper recruitment of melanosome biogenesis factors needed for the normal regulation of the size and number of RPE melanosomes. We previously proposed a model suggesting that Gαi3 controls the size of melanosomes in the RPE through the inhibition of vesicle trafficking from the trans-Golgi network (TGN) to the melanosomes [20]. This could explain the changes in RPE phenotype observed in ocular albinism when a non-functional OA1 protein (that cannot activate Gαi3) leads to a continuous vesicular traffic of membrane proteins to melanosomes resulting in the formation of macromelanosomes.…”

Section: Discussionmentioning

confidence: 99%

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A Constitutively Active Gαi3 Protein Corrects the Abnormal Retinal Pigment Epithelium Phenotype of Oa1−/− mice

et al. 2013

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PurposeOcular Albinism type 1 (OA1) is a disease caused by mutations in the OA1 gene and characterized by the presence of macromelanosomes in the retinal pigment epithelium (RPE) as well as abnormal crossing of the optic axons at the optic chiasm. We showed in our previous studies in mice that Oa1 activates specifically Gαi3 in its signaling pathway and thus, hypothesized that a constitutively active Gαi3 in the RPE of Oa1−/− mice might keep on the Oa1 signaling cascade and prevent the formation of macromelanosomes. To test this hypothesis, we have generated transgenic mice that carry the constitutively active Gαi3 (Q204L) protein in the RPE of Oa1−/− mice and are now reporting the effects that the transgene produced on the Oa1−/− RPE phenotype.MethodsTransgenic mice carrying RPE-specific expression of the constitutively active Gαi3 (Q204L) were generated by injecting fertilized eggs of Oa1−/− females with a lentivirus containing the Gαi3 (Q204L) cDNA. PCR, Southern blots, Western blots and confocal microscopy were used to confirm the presence of the transgene in the RPE of positive transgenic mice. Morphometrical analyses were performed using electron microscopy to compare the size and number of melanosomes per RPE area in putative Oa1−/−, Gαi3 (Q204L) transgenic mice with those of wild-type NCrl and Oa1−/− mice.ResultsWe found a correlation between the presence of the constitutively active Gαi3 (Q204L) transgene and the rescue of the normal phenotype of RPE melanosomes in Oa1−/−, Gαi3 (Q204L) mice. These mice have higher density of melanosomes per RPE area and a larger number of small melanosomes than Oa1−/− mice, and their RPE phenotype is similar to that of wild-type mice.ConclusionsOur results show that a constitutively active Gαi3 protein can by-pass the lack of Oa1 protein in Oa1−/− mice and consequently rescue the RPE melanosomal phenotype.

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“…The total retinal RNA from healthy human donor eyes that were obtained from the National Disease Research Interchange (Philadelphia, PA, USA) was generously provided by Dr. D. B. Farber (UCLA; Young et al 2011). The RNA was DNase treated with Turbo DNA-free (Ambion, Austin, TX, USA) and purified with RNeasy MiniElute Cleanup kit (Qiagen, Valencia, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

RNA-binding protein Rbpms is represented in human retinas by isoforms A and C and its transcriptional regulation involves Sp1-binding site

Piri

2018

Mol Genet Genomics

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Rbpms (RNA-binding protein with multiple splicing) is a member of the RRM (RNA Recognition Motif) family of RNA-binding proteins, which is expressed as multiple alternatively spliced transcripts encoding different protein isoforms. We have shown earlier that Rbpms expression in the retina is restricted to retinal ganglion cells (RGCs), and have characterized this gene as a marker for RGCs. The aim of this study was to identify isoforms representing Rbpms in human retinas and to analyze its transcriptional regulation. We found that Rbpms is expressed as transcription variants 1 and 3 encoding isoforms A and C, respectively. These isoforms are encoded by the same first 6 exons but have different C-terminal ends encoded by exon 8 in variant 1 and exon 7 in variant 3. Computational analysis of the Rbpms 5′ untranslated and flanking regions reveals the presence of three CpG islands and four predicted promoter regions (PPRs). The effect of PPR 1 (− 1672/− 1420) and PPR2 (− 330/− 79) on transcriptional activation was minimal, whereas PPR 3 (− 73/+ 177) and PPR4 (+ 274/+ 524) induced the expression by ~ 7 and ninefold compared to control, respectively. The maximum activity, a 30-fold increase above the control level, was obtained from the construct containing both PPRs 3 and 4. Site-directed mutagenesis of several cis-elements within PPR3 and PPR4 including five for Sp1, one for AP1, and two for NF-kB showed that mutation of the first three and especially the first GC box resulted in a threefold downregulation of gene expression. AP1, NF-kB, and two downstream Sp1 sites had no significant effect on expression level. The possible involvement of the GC box 1 at position − 54 in transcriptional regulation of Rbpms was corroborated by EMSA, which showed formation of a DNA–protein complex in the presence of the oligonucleotide corresponding to this Sp1-binding site.

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Specific Interaction of Gαi3 with the Oa1 G-Protein Coupled Receptor Controls the Size and Density of Melanosomes in Retinal Pigment Epithelium

Cited by 20 publications

References 32 publications

Autoregulatory circuit regulating basolateral cargo export from the TGN: role of the orphan receptor GPRC5A in PKD signaling and cell polarity

Autoregulatory circuit regulating basolateral cargo export from the TGN: role of the orphan receptor GPRC5A in PKD signaling and cell polarity

A Constitutively Active Gαi3 Protein Corrects the Abnormal Retinal Pigment Epithelium Phenotype of Oa1−/− mice

RNA-binding protein Rbpms is represented in human retinas by isoforms A and C and its transcriptional regulation involves Sp1-binding site

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