RESUMEN
AXL, a TAM receptor tyrosine kinase (RTK), and its ligand growth arrest-specific 6 (GAS6) are implicated in cancer metastasis and drug resistance, and cellular entry of viruses. Given this, AXL is an attractive therapeutic target, and its inhibitors are being tested in cancer and COVID-19 clinical trials. Still, astonishingly little is known about intracellular mechanisms that control its function. Here, we characterized endocytosis of AXL, a process known to regulate intracellular functions of RTKs. Consistent with the notion that AXL is a primary receptor for GAS6, its depletion was sufficient to block GAS6 internalization. We discovered that upon receptor ligation, GAS6-AXL complexes were rapidly internalized via several endocytic pathways including both clathrin-mediated and clathrin-independent routes, among the latter the CLIC/GEEC pathway and macropinocytosis. The internalization of AXL was strictly dependent on its kinase activity. In comparison to other RTKs, AXL was endocytosed faster and the majority of the internalized receptor was not degraded but rather recycled via SNX1-positive endosomes. This trafficking pattern coincided with sustained AKT activation upon GAS6 stimulation. Specifically, reduced internalization of GAS6-AXL upon the CLIC/GEEC downregulation intensified, whereas impaired recycling due to depletion of SNX1 and SNX2 attenuated AKT signaling. Altogether, our data uncover the coupling between AXL endocytic trafficking and AKT signaling upon GAS6 stimulation. Moreover, our study provides a rationale for pharmacological inhibition of AXL in antiviral therapy as viruses utilize GAS6-AXL-triggered endocytosis to enter cells.
Asunto(s)
Endocitosis , Péptidos y Proteínas de Señalización Intercelular , Proteínas Proto-Oncogénicas , Proteínas Tirosina Quinasas Receptoras , Antivirales/farmacología , Antivirales/uso terapéutico , COVID-19/metabolismo , COVID-19/terapia , Clatrina/metabolismo , Clatrina/fisiología , Endocitosis/efectos de los fármacos , Endocitosis/genética , Endocitosis/fisiología , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/fisiología , Neoplasias/metabolismo , Neoplasias/terapia , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-akt/fisiología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/fisiología , Tirosina Quinasa del Receptor AxlRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, so understanding its biology and infection mechanisms is critical to facing this major medical challenge. SARS-CoV-2 is known to use its spike glycoprotein to interact with the cell surface as a first step in the infection process. As for other coronaviruses, it is likely that SARS-CoV-2 next undergoes endocytosis, but whether or not this is required for infectivity and the precise endocytic mechanism used are unknown. Using purified spike glycoprotein and lentivirus pseudotyped with spike glycoprotein, a common model of SARS-CoV-2 infectivity, we now demonstrate that after engagement with the plasma membrane, SARS-CoV-2 undergoes rapid, clathrin-mediated endocytosis. This suggests that transfer of viral RNA to the cell cytosol occurs from the lumen of the endosomal system. Importantly, we further demonstrate that knockdown of clathrin heavy chain, which blocks clathrin-mediated endocytosis, reduces viral infectivity. These discoveries reveal that SARS-CoV-2 uses clathrin-mediated endocytosis to gain access into cells and suggests that this process is a key aspect of virus infectivity.