Cooperation of the IFT-A complex with the IFT-B complex is required for ciliary retrograde protein trafficking and GPCR import

Little is known about how the IFT-A and IFT-B complexes of the intraflagellar transport machinery cooperate to mediate ciliary protein trafficking. We show that IFT144-IFT122 and IFT88-IFT52-IFT46 contribute to the IFT-A-IFT-B interface and that the interaction of IFT-A with IFT-B is required for retrograde trafficking of the IFT machinery and ciliary entry of G protein-coupled receptors.

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Intraflagellar transport-A complex mediates ciliary entry and retrograde trafficking of ciliary G protein–coupled receptors

Molecular Biology of the Cell ◽

10.1091/mbc.e16-11-0813 ◽

2017 ◽

Vol 28 (3) ◽

pp. 429-439 ◽

Cited By ~ 55

Author(s):

Tomoaki Hirano ◽

Yohei Katoh ◽

Kazuhisa Nakayama

Keyword(s):

G Protein ◽

Protein Interactions ◽

Protein Trafficking ◽

Intraflagellar Transport ◽

G Protein Coupled Receptors ◽

Protein Protein Interactions ◽

Retrograde Trafficking ◽

The Core ◽

Ciliary Protein ◽

G Protein Coupled

Cilia serve as cellular antennae where proteins involved in sensory and developmental signaling, including G protein–coupled receptors (GPCRs), are specifically localized. Intraflagellar transport (IFT)-A and -B complexes mediate retrograde and anterograde ciliary protein trafficking, respectively. Using a visible immunoprecipitation assay to detect protein–protein interactions, we show that the IFT-A complex is divided into a core subcomplex, composed of IFT122/IFT140/IFT144, which is associated with TULP3, and a peripheral subcomplex, composed of IFT43/IFT121/IFT139, where IFT139 is most distally located. IFT139-knockout (KO) and IFT144-KO cells demonstrated distinct phenotypes: IFT139-KO cells showed the accumulation of IFT-A, IFT-B, and GPCRs, including Smoothened and GPR161, at the bulged ciliary tips; IFT144-KO cells showed failed ciliary entry of IFT-A and GPCRs and IFT-B accumulation at the bulged tips. These observations demonstrate the distinct roles of the core and peripheral IFT-A subunits: IFT139 is dispensable for IFT-A assembly but essential for retrograde trafficking of IFT-A, IFT-B, and GPCRs; in contrast, IFT144 is essential for functional IFT-A assembly and ciliary entry of GPCRs but dispensable for anterograde IFT-B trafficking. Thus the data presented here demonstrate that the IFT-A complex mediates not only retrograde trafficking but also entry into cilia of GPCRs.

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Modulation of proteostasis and protein trafficking: a therapeutic avenue for misfolded G protein-coupled receptors causing disease in humans

Emerging Topics in Life Sciences ◽

10.1042/etls20180055 ◽

2019 ◽

Vol 3 (1) ◽

pp. 39-52

Author(s):

Alfredo Ulloa-Aguirre ◽

Jo Ann Janovick

Keyword(s):

G Protein ◽

Protein Trafficking ◽

Protein Misfolding ◽

Hypogonadotropic Hypogonadism ◽

Three Dimensional ◽

G Protein Coupled Receptors ◽

Dimensional Structure ◽

Stable Conformation ◽

Mutant Proteins ◽

G Protein Coupled

Abstract Proteostasis refers to the process whereby the cell maintains in equilibrium the protein content of different compartments. This system consists of a highly interconnected network intended to efficiently regulate the synthesis, folding, trafficking, and degradation of newly synthesized proteins. Molecular chaperones are key players of the proteostasis network. These proteins assist in the assembly and folding processes of newly synthesized proteins in a concerted manner to achieve a three-dimensional structure compatible with export from the endoplasmic reticulum to other cell compartments. Pharmacologic interventions intended to modulate the proteostasis network and tackle the devastating effects of conformational diseases caused by protein misfolding are under development. These include small molecules called pharmacoperones, which are highly specific toward the target protein serving as a molecular framework to cause misfolded mutant proteins to fold and adopt a stable conformation suitable for passing the scrutiny of the quality control system and reach its correct location within the cell. Here, we review the main components of the proteostasis network and how pharmacoperones may be employed to correct misfolding of two G protein-coupled receptors, the vasopressin 2 receptor and the gonadotropin-releasing hormone receptor, whose mutations lead to X-linked nephrogenic diabetes insipidus and congenital hypogonadotropic hypogonadism in humans respectively.

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The Ciliary Lumen Accommodates Passive Diffusion and Vesicle Trafficking in Cytoplasmic-Ciliary Transport

10.1101/704213 ◽

2019 ◽

Cited By ~ 1

Author(s):

Andrew Ruba ◽

Wangxi Luo ◽

Jingjie Yu ◽

Daisuke Takao ◽

Athanasios Evangelou ◽

...

Keyword(s):

Chlamydomonas Reinhardtii ◽

G Protein ◽

Single Molecule ◽

Primary Cilium ◽

Intraflagellar Transport ◽

Vesicle Transport ◽

Passive Diffusion ◽

G Protein Coupled Receptors ◽

Live Cells ◽

G Protein Coupled

AbstractTransport of membrane and cytosolic proteins into the primary cilium is essential for its role in cellular signaling. Using single molecule microscopy, we mapped the movement of membrane and soluble proteins at the base of the primary cilium. In addition to the well-known intraflagellar transport (IFT) route, we identified two new pathways within the lumen of the primary cilium - passive diffusional and vesicle transport routes - that are adopted by proteins for cytoplasmic-cilium transport in live cells. Independent of the IFT path, approximately half of IFT motors (KIF3A) and cargo (α-tubulin) take the passive diffusion route and more than half of membrane-embedded G protein coupled receptors (SSTR3 and HTR6) use RAB8A-regulated vesicles to transport into and inside cilia. Furthermore, ciliary lumen transport is the preferred route for membrane proteins in the early stages of ciliogenesis and inhibition of SSTR3 vesicle transport completely blocks ciliogenesis. Furthermore, clathrin-mediated, signal-dependent internalization of SSTR3 also occurs through the ciliary lumen. These transport routes were also observed in Chlamydomonas reinhardtii flagella, suggesting their conserved roles in trafficking of ciliary proteins.

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Anterograde trafficking of ciliary MAP kinase–like ICK/CILK1 by the intraflagellar transport machinery is required for intraciliary retrograde protein trafficking

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014142 ◽

2020 ◽

Vol 295 (38) ◽

pp. 13363-13376 ◽

Cited By ~ 1

Author(s):

Kentaro Nakamura ◽

Tatsuro Noguchi ◽

Mariko Takahara ◽

Yoshihiro Omori ◽

Takahisa Furukawa ◽

...

Keyword(s):

Protein Trafficking ◽

Intraflagellar Transport ◽

Extracellular Vesicle ◽

Mitogen Activated Protein Kinase ◽

Ciliary Protein ◽

Mitogen Activated Protein ◽

Exogenous Expression ◽

Bidirectional Movement ◽

Dependent Transport ◽

G Protein Coupled

ICK (also known as CILK1) is a mitogen-activated protein kinase–like kinase localized at the ciliary tip. Its deficiency is known to result in the elongation of cilia and causes ciliopathies in humans. However, little is known about how ICK is transported to the ciliary tip. We here show that the C-terminal noncatalytic region of ICK interacts with the intraflagellar transport (IFT)–B complex of the IFT machinery and participates in its transport to the ciliary tip. Furthermore, total internal reflection fluorescence microscopy demonstrated that ICK undergoes bidirectional movement within cilia, similarly to IFT particles. Analysis of ICK knockout cells demonstrated that ICK deficiency severely impairs the retrograde trafficking of IFT particles and ciliary G protein–coupled receptors. In addition, we found that in ICK knockout cells, ciliary proteins are accumulated at the bulged ciliary tip, which appeared to be torn off and released into the environment as an extracellular vesicle. The exogenous expression of various ICK constructs in ICK knockout cells indicated that the IFT-dependent transport of ICK, as well as its kinase activity and phosphorylation at the canonical TDY motif, is essential for ICK function. Thus, we unequivocally show that ICK transported to the ciliary tip is required for retrograde ciliary protein trafficking and consequently for normal ciliary function.

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