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Bardet-Biedl syndrome-associated small GTPase ARL6 (BBS3) functions at or near the ciliary gate and modulates Wnt signaling.

Wiens CJ, Tong Y, Esmail MA, Oh E, Gerdes JM, Wang J, Tempel W, Rattner JB, Katsanis N, Park HW, Leroux MR - J. Biol. Chem. (2010)

Bottom Line: The expansive family of metazoan ADP-ribosylation factor and ADP-ribosylation factor-like small GTPases is known to play essential roles in modulating membrane trafficking and cytoskeletal functions.Here, we present the crystal structure of ARL6, mutations in which cause Bardet-Biedl syndrome (BBS3), and reveal its unique ring-like localization at the distal end of basal bodies, in proximity to the so-called ciliary gate where vesicles carrying ciliary cargo fuse with the membrane.Importantly, this signaling function is lost in ARL6 variants containing BBS-associated point mutations.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.

ABSTRACT
The expansive family of metazoan ADP-ribosylation factor and ADP-ribosylation factor-like small GTPases is known to play essential roles in modulating membrane trafficking and cytoskeletal functions. Here, we present the crystal structure of ARL6, mutations in which cause Bardet-Biedl syndrome (BBS3), and reveal its unique ring-like localization at the distal end of basal bodies, in proximity to the so-called ciliary gate where vesicles carrying ciliary cargo fuse with the membrane. Overproduction of GDP- or GTP-locked variants of ARL6/BBS3 in vivo influences primary cilium length and abundance. ARL6/BBS3 also modulates Wnt signaling, a signal transduction pathway whose association with cilia in vertebrates is just emerging. Importantly, this signaling function is lost in ARL6 variants containing BBS-associated point mutations. By determining the structure of GTP-bound ARL6/BBS3, coupled with functional assays, we provide a mechanistic explanation for such pathogenic alterations, namely altered nucleotide binding. Our findings therefore establish a previously unknown role for ARL6/BBS3 in mammalian ciliary (dis)assembly and Wnt signaling and provide the first structural information for a BBS protein.

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Overexpression of wild-type but not mutant forms of ARL6/BBS3 alters responsiveness to Wnt3a ligand. In hTERT-RPE TOPflash reporter cells, overexpression of ARL6/BBS3 leads to increased β-catenin activity in response to Wnt3a ligand but no global dysregulation of β-catenin signaling. In contrast, overexpression of ARL6(T31R) and ARL6(Q73L) has no marked effect on β-catenin transcriptional activity. Cells are consistently 70–80% confluent and ciliated as observed by staining with an anti-acetylated tubulin antibody (data not shown). Error bars represent standard deviation; only the BBS3 wild type (WT) samples are statistically different from the empty vector control (p < 0.05).
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Figure 7: Overexpression of wild-type but not mutant forms of ARL6/BBS3 alters responsiveness to Wnt3a ligand. In hTERT-RPE TOPflash reporter cells, overexpression of ARL6/BBS3 leads to increased β-catenin activity in response to Wnt3a ligand but no global dysregulation of β-catenin signaling. In contrast, overexpression of ARL6(T31R) and ARL6(Q73L) has no marked effect on β-catenin transcriptional activity. Cells are consistently 70–80% confluent and ciliated as observed by staining with an anti-acetylated tubulin antibody (data not shown). Error bars represent standard deviation; only the BBS3 wild type (WT) samples are statistically different from the empty vector control (p < 0.05).

Mentions: To explore the potential role of ARL6/BBS3 in Wnt signaling, we expressed ARL6/BBS3 in an hTERT-RPE1 cell line transfected with a pTOPflash reporter plasmid (Fig. 7) and a stable HEK293T Wnt reporter cell line (supplemental Fig. 4A). Interestingly, the effect of ARL6/BBS3 on β-catenin activity is dosage-dependent, because overexpression leads to an approximate 2- or 6-fold increased Wnt3a response in the hTERT-RPE1 (Fig. 7) and HEK293T (supplemental Fig. 4A) cell line compared to wild-type cells, respectively. In contrast, overexpression of ARL6(T31R) and ARL6(Q73L), now known to cause ciliary anomalies (Fig. 6), did not have an effect on β-catenin transcriptional activity (Fig. 7). Although ARL6(T31R) represents a pathogenic mutation, ARL6(Q73L) is not a mutation identified in BBS3 patients. Therefore, to further investigate whether or not Wnt signaling may be abrogated in BBS patients, we tested the response to Wnt3a ligand in additional overexpressed ARL6 pathogenic variants, namely ARL6(G169A) and ARL6(L170W). Similarly to ARL6(T31R) and ARL6(Q73L), we observed a loss of responsivity to the Wnt3a signal and β-catenin transcriptional activity compared to when wild-type ARL6 is overexpressed (supplemental Fig. 4B). Together, these results demonstrate that ARL6/BBS3 can modulate Wnt signaling in mammalian cells and that its effect may be different from that of other BBS proteins (see below).


Bardet-Biedl syndrome-associated small GTPase ARL6 (BBS3) functions at or near the ciliary gate and modulates Wnt signaling.

Wiens CJ, Tong Y, Esmail MA, Oh E, Gerdes JM, Wang J, Tempel W, Rattner JB, Katsanis N, Park HW, Leroux MR - J. Biol. Chem. (2010)

Overexpression of wild-type but not mutant forms of ARL6/BBS3 alters responsiveness to Wnt3a ligand. In hTERT-RPE TOPflash reporter cells, overexpression of ARL6/BBS3 leads to increased β-catenin activity in response to Wnt3a ligand but no global dysregulation of β-catenin signaling. In contrast, overexpression of ARL6(T31R) and ARL6(Q73L) has no marked effect on β-catenin transcriptional activity. Cells are consistently 70–80% confluent and ciliated as observed by staining with an anti-acetylated tubulin antibody (data not shown). Error bars represent standard deviation; only the BBS3 wild type (WT) samples are statistically different from the empty vector control (p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2871489&req=5

Figure 7: Overexpression of wild-type but not mutant forms of ARL6/BBS3 alters responsiveness to Wnt3a ligand. In hTERT-RPE TOPflash reporter cells, overexpression of ARL6/BBS3 leads to increased β-catenin activity in response to Wnt3a ligand but no global dysregulation of β-catenin signaling. In contrast, overexpression of ARL6(T31R) and ARL6(Q73L) has no marked effect on β-catenin transcriptional activity. Cells are consistently 70–80% confluent and ciliated as observed by staining with an anti-acetylated tubulin antibody (data not shown). Error bars represent standard deviation; only the BBS3 wild type (WT) samples are statistically different from the empty vector control (p < 0.05).
Mentions: To explore the potential role of ARL6/BBS3 in Wnt signaling, we expressed ARL6/BBS3 in an hTERT-RPE1 cell line transfected with a pTOPflash reporter plasmid (Fig. 7) and a stable HEK293T Wnt reporter cell line (supplemental Fig. 4A). Interestingly, the effect of ARL6/BBS3 on β-catenin activity is dosage-dependent, because overexpression leads to an approximate 2- or 6-fold increased Wnt3a response in the hTERT-RPE1 (Fig. 7) and HEK293T (supplemental Fig. 4A) cell line compared to wild-type cells, respectively. In contrast, overexpression of ARL6(T31R) and ARL6(Q73L), now known to cause ciliary anomalies (Fig. 6), did not have an effect on β-catenin transcriptional activity (Fig. 7). Although ARL6(T31R) represents a pathogenic mutation, ARL6(Q73L) is not a mutation identified in BBS3 patients. Therefore, to further investigate whether or not Wnt signaling may be abrogated in BBS patients, we tested the response to Wnt3a ligand in additional overexpressed ARL6 pathogenic variants, namely ARL6(G169A) and ARL6(L170W). Similarly to ARL6(T31R) and ARL6(Q73L), we observed a loss of responsivity to the Wnt3a signal and β-catenin transcriptional activity compared to when wild-type ARL6 is overexpressed (supplemental Fig. 4B). Together, these results demonstrate that ARL6/BBS3 can modulate Wnt signaling in mammalian cells and that its effect may be different from that of other BBS proteins (see below).

Bottom Line: The expansive family of metazoan ADP-ribosylation factor and ADP-ribosylation factor-like small GTPases is known to play essential roles in modulating membrane trafficking and cytoskeletal functions.Here, we present the crystal structure of ARL6, mutations in which cause Bardet-Biedl syndrome (BBS3), and reveal its unique ring-like localization at the distal end of basal bodies, in proximity to the so-called ciliary gate where vesicles carrying ciliary cargo fuse with the membrane.Importantly, this signaling function is lost in ARL6 variants containing BBS-associated point mutations.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.

ABSTRACT
The expansive family of metazoan ADP-ribosylation factor and ADP-ribosylation factor-like small GTPases is known to play essential roles in modulating membrane trafficking and cytoskeletal functions. Here, we present the crystal structure of ARL6, mutations in which cause Bardet-Biedl syndrome (BBS3), and reveal its unique ring-like localization at the distal end of basal bodies, in proximity to the so-called ciliary gate where vesicles carrying ciliary cargo fuse with the membrane. Overproduction of GDP- or GTP-locked variants of ARL6/BBS3 in vivo influences primary cilium length and abundance. ARL6/BBS3 also modulates Wnt signaling, a signal transduction pathway whose association with cilia in vertebrates is just emerging. Importantly, this signaling function is lost in ARL6 variants containing BBS-associated point mutations. By determining the structure of GTP-bound ARL6/BBS3, coupled with functional assays, we provide a mechanistic explanation for such pathogenic alterations, namely altered nucleotide binding. Our findings therefore establish a previously unknown role for ARL6/BBS3 in mammalian ciliary (dis)assembly and Wnt signaling and provide the first structural information for a BBS protein.

Show MeSH
Related in: MedlinePlus