TRAIL-death receptor endocytosis and apoptosis are selectively regulated by dynamin-1 activation Carlos R. Reisa, Ping-Hung Chena, Nawal Bendrisa, and Sandra L. Schmida,1 aDepartment of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390 Edited by Ira Mellman, Genentech, Inc., South San Francisco, CA, and approved December 5, 2016 (received for review September 8, 2016) Clathrin-mediated endocytosis (CME) constitutes the major path- high curvature-generating properties when compared with Dyn2, way for uptake of signaling receptors into eukaryotic cells. As making it more suited for rapid compensatory endocytosis (13). such, CME regulates signaling from cell-surface receptors, but Hence, at the synapse, Dyn1 is well-suited to mediate rapid whether and how specific signaling receptors reciprocally regulate compensatory endocytosis and synaptic vesicle recycling following the CME machinery remains an open question. Although best neurotransmission (14). Unexpectedly, we have recently shown studied for its role in membrane fission, the GTPase dynamin also that Dyn1 can also be activated in nonneuronal cells, downstream regulates early stages of CME. We recently reported that dynamin-1 of an Akt/GSK3β signaling cascade to alter the rate and regulation (Dyn1), previously assumed to be neuron-specific, can be selectively of CME (15), linking endocytosis to signaling. activated in cancer cells to alter endocytic trafficking. Here we Activation of the apoptosis-signaling machinery by TRAIL report that dynamin isoforms differentially regulate the endocyto- (TNF-related apoptosis-inducing ligand) through the engage- sis and apoptotic signaling downstream of TNF-related apoptosis- inducing ligand–death receptor (TRAIL–DR) complexes in several ment of death receptors (DRs) has gained considerable interest – cancer cells. Whereas the CME of constitutively internalized trans- as a potential anticancer strategy (16 19); however, its efficacy is ferrin receptors is mainly dependent on the ubiquitously expressed limited by a variety of cancer cell-resistance mechanisms. TRAIL Dyn2, TRAIL-induced DR endocytosis is selectively regulated by ac- binding to its cognate death receptors (DR4 and DR5) triggers tivation of Dyn1. We show that TRAIL stimulation activates ryano- the formation of the death-inducing signaling complex by the dine receptor-mediated calcium release from endoplasmic reticulum recruitment of the adaptor FADD (Fas-associated death domain) CELL BIOLOGY stores, leading to calcineurin-mediated dephosphorylation and ac- and the initiator caspase-8 (20). Activated caspase-8 can then ac- tivation of Dyn1, TRAIL–DR endocytosis, and increased resistance to tivate effector caspase-3 and -7, leading to cell-extrinsic apoptosis. TRAIL-induced apoptosis. TRAIL–DR-mediated ryanodine receptor Binding of human recombinant TRAIL to its cognate apoptosis- activation and endocytosis is dependent on early caspase-8 activa- inducing DR (DR4 or DR5) stimulates their internalization via tion. These findings delineate specific mechanisms for the reciprocal CME (21, 22). However, there are conflicting reports as to the crosstalk between signaling and the regulation of CME, leading to effect of TRAIL-induced endocytosis of DRs on apoptotic sig- autoregulation of endocytosis and signaling downstream of surface naling (21–23). Initial experimental data suggested that CME of receptors. TRAIL–DR complexes has an inhibitory effect on TRAIL-induced apoptosis (21, 22). However, using overexpressed dominant-nega- clathrin-mediated endocytosis | calcineurin | ryanodine receptor | programmed cell death | caspases tive dynamins to block CME, others have shown that endocytosis of TRAIL–DR is required for apoptosis, and proposed possible cell-type–specific differences in TRAIL signaling (23). eceptor-mediated endocytosis plays a critical role in regulat- Ring signaling, by either promoting rapid endocytosis of ligand– receptor complexes and attenuating cell-surface signaling, or by Significance promoting the formation of endosomes that can serve as signaling platforms for these complexes (1, 2). Clathrin-mediated endocy- Clathrin-mediated endocytosis (CME) regulates receptor traf- tosis (CME) is one of the most important and well-characterized ficking, thereby affecting several cellular signaling pathways. endocytic pathways in eukaryotes (3, 4). The CME core compo- We discovered that dynamin-1 is selectively activated down- nents—clathrin, dynamin, and adaptor protein 2 (AP2)—interact stream of TNF-related apoptosis-inducing ligand–death recep- with several endocytic accessory proteins to initiate, stabilize, and tors (TRAIL–DRs) to self-regulate their endocytosis, attenuate promote the maturation of clathrin-coated pits (CCPs). Following apoptotic signaling, and increase cell survival. Activation of 2+ maturation, CCP scission is catalyzed by the large GTPase dyna- initiator caspase-8 by TRAIL–DRs triggers spikes of Ca min, leading to the formation of cargo-containing vesicles (5, 6). through ryanodine receptor calcium channels, activating calci- Once thought to be a constitutive process, it is now recognized neurin, and in turn dephosphorylating dynamin-1 to promote – that CME can be highly regulated (7), but many questions remain cargo-selective endocytosis of TRAIL DR. This study delineates as to the molecular mechanisms underlying the regulation of specific mechanisms linking signaling downstream of cell-sur- CME. Moreover, recent data have suggested that signaling G face receptors to the regulation of cargo-selective CME, and protein-coupled receptors (GPCRs) can directly regulate CCP thus their signaling properties. Cancer cell-specific adaptation dynamics through selective recruitment of dynamin and endocytic of this bidirectional crosstalk between signaling and CME has accessory proteins (8, 9). The extent of possible crosstalk between implications for tumor progression and metastasis. signaling receptors and CME has not been explored. Author contributions: C.R.R. and S.L.S. designed research; C.R.R., P.-H.C., and N.B. per- Dynamins are master regulators of CME. In addition to their formed research; C.R.R. contributed new reagents/analytic tools; C.R.R., P.-H.C., and N.B. role in promoting the fission of invaginated CCPs, dynamins analyzed data; and C.R.R. and S.L.S. wrote the paper. control earlier rate-limiting steps of clathrin-coated vesicle for- The authors declare no conflict of interest. – mation (10 12). There are three dynamin isoforms in vertebrates: This article is a PNAS Direct Submission. dynamin-1 (Dyn1) is predominantly expressed in neurons, dyna- 1To whom correspondence should be addressed. Email: sandra.schmid@utsouthwestern. min-2 (Dyn2) is ubiquitously expressed, and dynamin-3 (Dyn3) is edu. expressed in neurons, lung, and testis. Dyn1 and Dyn2 are distinct This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. in their curvature sensing/generating properties: Dyn1 exhibits 1073/pnas.1615072114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1615072114 PNAS Early Edition | 1of6 Downloaded by guest on September 23, 2021 In this study, we sought to determine the contribution of CME AP2 or CHC (Fig. 1A and Fig. S1 A and B). This finding was true to the regulation of signaling via TRAIL–DR. Experimental for both DR4- and DR5-triggered apoptosis, as assessed using down-regulation of all of the core components of the CME DR-selective variants of TRAIL (24, 25) (Fig. S1 C and D). machinery revealed that dynamin isoforms differentially regulate Western blotting confirmed that the differential effects of Dyn1 CME of selected cargoes: whereas CME of constitutively in- and Dyn2 were not a result of differential efficiencies of ternalized transferrin receptors (TfnR) requires Dyn2, CME of knockdown or because of compensatory up-regulation of the TRAIL–DR complexes is Dyn1-dependent. We discovered that nontargeted isoform (Fig. S2 A and B). Indeed, depletion of TRAIL-induced early activation of caspase-8 results in ryano- Dyn1 did not impact the protein levels of Dyn2 in A549 cells and + dine receptor (RyR)-mediated calcium release, causing the Ca2 MDA-MB-231 cells and Dyn2 knockdown only moderately (by and calcineurin-dependent activation of Dyn1, Dyn1-dependent ∼20%) increased the protein levels of Dyn1 in A549 cells (Fig. CME of TRAIL–DR, and suppression of TRAIL-induced S2 A and B). apoptosis. To determine whether the isoform-specific functions of Dyn1 and Dyn2 were related to their roles in endocytosis, we assessed Results and Discussion the effect of their down-regulation on TRAIL uptake. siRNA- Dynamins Differentially Regulate Cargo-Selective Endocytosis and mediated depletion of Dyn1 potently reduced TRAIL–DR en- TRAIL-Induced Apoptosis. Given the existence of conflicting re- docytosis to the same extent as depletion of AP2 or CHC (Fig. ports (21–23), we directly tested the role of CME in regulating 1B), resulting in increased levels of surface-bound TRAIL (Fig. TRAIL-induced apoptosis by small-interfering RNA (siRNA)- S2 C and D). Depletion of Dyn2 was much less effective in re- mediated knockdown of core components of the CME machin- ducing TRAIL–DR endocytosis. The opposite was true for uptake ery, namely the coat protein clathrin heavy chain (CHC), the of the classic CME cargo, TfnR, which was mainly dependent on AP2, and the ubiquitously expressed isoform of dynamin, Dyn2. Dyn2 but not on Dyn1 (Fig. 1C). To confirm these effects, we TRAIL-sensitive MDA-MB-231 human