The gap junction complex functions as a transport channel across the membrane. Among gap junction subunits, gap junction protein α1 (GJA1) is the most commonly expressed subunit. A recent study showed that GJA1 is necessary for the maintenance of motile cilia; however, the molecular mechanism and function of GJA1 in ciliogenesis remain unknown. Here, we examined the functions of GJA1 during ciliogenesis in human retinal pigment epithelium-1 and Xenopus laevis embryonic multiciliated-cells. GJA1 localizes to the motile ciliary axonemes or pericentriolar regions beneath the primary cilium. GJA1 depletion caused malformation of both the primary cilium and motile cilia. Further study revealed that GJA1 depletion affected several ciliary proteins such as BBS4, CP110, and Rab11 in the pericentriolar region and basal body. Interestingly, CP110 removal from the mother centriole was significantly reduced by GJA1 depletion. Importantly, Rab11, a key regulator during ciliogenesis, was immunoprecipitated with GJA1, and GJA1 knockdown caused the mislocalization of Rab11. These findings suggest that GJA1 regulates ciliogenesis by interacting with the Rab11-Rab8 ciliary trafficking pathway.
All data generated or analysed during this study are included in the manuscript, supporting file and source data files
GJA1 IP/MS datasetDryad Digital Repository, doi:10.5061/dryad.tht76hdxt.
- Tae Joo Park
- Taejoon Kwon
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: All animal experiments were performed with appropriate ethical approval from the UNIST Institutional Animal Care and Use Committee (UNISTIACUC-19-22, UNISTIACUC-20-26).
- Gregory J Pazour, University of Massachusetts Medical School, United States
© 2022, Jang et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
β-Arrestins are master regulators of cellular signaling that operate by desensitizing ligand-activated G-protein-coupled receptors (GPCRs) at the plasma membrane and promoting their subsequent endocytosis. The endocytic activity of β-arrestins is ligand dependent, triggered by GPCR binding, and increasingly recognized to have a multitude of downstream signaling and trafficking consequences that are specifically programmed by the bound GPCR. However, only one biochemical ‘mode’ for GPCR-mediated triggering of the endocytic activity is presently known – displacement of the β-arrestin C-terminus (CT) to expose clathrin-coated pit-binding determinants that are masked in the inactive state. Here, we revise this view by uncovering a second mode of GPCR-triggered endocytic activity that is independent of the β-arrestin CT and, instead, requires the cytosolic base of the β-arrestin C-lobe (CLB). We further show each of the discrete endocytic modes is triggered in a receptor-specific manner, with GPCRs that bind β-arrestin transiently (‘class A’) primarily triggering the CLB-dependent mode and GPCRs that bind more stably (‘class B’) triggering both the CT and CLB-dependent modes in combination. Moreover, we show that different modes have opposing effects on the net signaling output of receptors – with the CLB-dependent mode promoting rapid signal desensitization and the CT-dependent mode enabling prolonged signaling. Together, these results fundamentally revise understanding of how β-arrestins operate as efficient endocytic adaptors while facilitating diversity and flexibility in the control of cell signaling.
The insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) control metabolic homeostasis and cell growth and proliferation. The IR and IGF1R form similar disulfide bonds linked homodimers in the apo-state; however, their ligand binding properties and the structures in the active state differ substantially. It has been proposed that the disulfide-linked C-terminal segment of α-chain (αCTs) of the IR and IGF1R control the cooperativity of ligand binding and regulate the receptor activation. Nevertheless, the molecular basis for the roles of disulfide-linked αCTs in IR and IGF1R activation are still unclear. Here, we report the cryo-EM structures of full-length mouse IGF1R/IGF1 and IR/insulin complexes with modified αCTs that have increased flexibility. Unlike the Γ-shaped asymmetric IGF1R dimer with a single IGF1 bound, the IGF1R with the enhanced flexibility of αCTs can form a T-shaped symmetric dimer with two IGF1s bound. Meanwhile, the IR with non-covalently linked αCTs predominantly adopts an asymmetric conformation with four insulins bound, which is distinct from the T-shaped symmetric IR. Using cell-based experiments, we further showed that both IGF1R and IR with the modified αCTs cannot activate the downstream signaling potently. Collectively, our studies demonstrate that the certain structural rigidity of disulfide-linked αCTs is critical for optimal IR and IGF1R signaling activation.