Page 3 - F. Cell biology

Tubby like protein family is an adaptor for
ciliary G protein-coupled receptor trafficking

Kyoungeun Kim¹ ¹, Seok Jun Moon¹ ¹
,
,
Oral Biology, Yonsei University College of Dentistry, Seoul 03722, Republic of Korea

Introduction

Cilia are highly specialized antennae-like cellular organelles serves as
mediator for cell signaling cascade. Dysfunction of cilia due to diverse factor
leads to clinical phenotype such as retinal degeneration, polycystic kidney
disease and obesity. An elaborate mechanism for establishing and
maintaining the compartmentalization of cilia is necessary for proper ciliary
function, which includes the entry, localization, and exit of specific signaling
molecules to and from the ciliary compartment.
In the previous study, TULP3 is known as adapters for the ciliary trafficking
of G protein-coupled receptors (GPCRs). Since Tubby like protein family
(TULP) shares N-terminal IFT binding domain and C-terminal Tubby domain,
it is intriguing to speculate that all TULPs function as adaptors for ciliary
membrane cargo trafficking. This study investigate whether TULPs regulate
ciliary protein trafficking. hTERT RPE-1(RPE1) cell is generally known for cell
line that doesn’t express TUB, we additionally identified TULP1 and TULP2
are not expressed in RPE1cell and used CRISPR/Cas9 system to generate
Tulp3 knockout cell, namely TULPs abolished cell. Subsequently, the primary Fig. 2. Ciliary formation of TULP3 knockout cell
cilia of wild type cell, Tulp3 knockout cells and each TULP rescued Tulp3
knockout cells were visualized by immunocytochemistry test. Ciliary formation
Tulp3 knockout cells were downregulated comparing to wild type cells and
length of cilia was also unambiguously shorter than wild type cells.
Interestingly, only TULP3 was able to fully rescue ciliary length and frequency
in Tulp3 knockout cells. TULP1, TULP2 and TULP3 were not able to rescue
ciliary length or formation.
Next, candidate ciliary GPCRs selected based on previous data were
transfected to Tulp3 knockout cells. Ciliary GPCRs which failed to localize
cilia in Tulp3 knockout cells tend to recover ciliary trafficking depending on
TULP1, TULP2, TULP3 and TUB. This result indicates TULPs are functionally
redundant in ciliary GPCR trafficking but selectively involved in maintenance
of primary cilium. Further, regarding the distinct tissue distribution between
TULPs, TULPs might have tissue-specific functions. This result indicates that
TULPs share similar functions but also might have functional diversity through
its differential tissue distribution.

Materials and Methods Fig. 3. Effect of TULPs in ciliary GPCRs trafficking.

Conclusions

• TULP3 serve as core factor that regulate primary cilia
development among TULPs.
• TULP1, TULP2, TULP3 and TUB function as adaptor for ciliary
molecule trafficking.

References

• Badgandi, H. B., Hwang, S. H., Shimada, I. S., Loriot, E., & Mukhopadhyay, S.
(2017). Tubby family proteins are adapters for ciliary trafficking of integral
membrane proteins. Journal of Cell Biology, 216(3), 743-760.
Results • Han, S., Miyoshi, K., Shikada, S., Amano, G., Wang, Y., Yoshimura, T., &
Katayama, T. (2019). TULP3 is required for localization of membrane-
associated proteins ARL13B and INPP5E to primary cilia. Biochemical and
biophysical research communications, 509(1), 227-234.
• Loktev, A. V., & Jackson, P. K. (2013). Neuropeptide Y family receptors traffic
via the Bardet-Biedl syndrome pathway to signal in neuronal primary cilia. Cell
reports, 5(5), 1316-1329.
• Omori, Y., Chaya, T., Yoshida, S., Irie, S., Tsujii, T., & Furukawa, T. (2015).
Identification of G protein-coupled receptors (GPCRs) in primary cilia and their
possible involvement in body weight control. PLoS One, 10(6).

Acknowledgments

This work was supported by the BK21 plus program through
the National Research Foundation (NRF) funded by the
Ministry of Education of Korea.
Fig. 1. CRISPR/Cas9-mediated generation of Tulp3 knockout