Maria N Balatskaya1, Georgy V Sharonov2, Alexandra I Baglay3, Yury P Rubtsov2, Vsevolod A Tkachuk3. 1. Faculty of Medicine, Lomonosov Moscow State University, Lomonosovskiy av. 27-1, 119192 Moscow, Russia. Electronic address: m.balatskaya@fbm.msu.ru. 2. Faculty of Medicine, Lomonosov Moscow State University, Lomonosovskiy av. 27-1, 119192 Moscow, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Miklukho-Maklaya str. 16/10, 117997 Moscow, Russia. 3. Faculty of Medicine, Lomonosov Moscow State University, Lomonosovskiy av. 27-1, 119192 Moscow, Russia.
Abstract
BACKGROUND: Unlike other cadherins, T-cadherin does not mediate strong cell-cell adhesion. It has two soluble ligands: low density lipoprotein (LDL) and high-molecular-weight (HMW) adiponectin. LDL binding to T-cadherin induces calcium signaling, migration, and proliferation, and has proatherogenic effects, but adiponectin binding promotes antiatherogenic effects. The reasons for this difference and mechanism of signal transduction by glycosylphosphatidylinositol (GPI)-anchored T-cadherin are unknown. METHODS: We compared the ability of LDL and HMW adiponectin to induce calcium signaling, T-cadherin clustering and internalization. We measured calcium signaling in smooth muscle cells and T-cadherin expressing HEK293 using single-cell imaging. To study receptor clustering, we tested three different T-cadherin labeling strategies and then utilized confocal microscopy and flow cytometry assays based on Förster resonance energy transfer (FRET). RESULTS: Enzymatically labeled T-cadherin retained its cellular localization and physiological activity, features that were otherwise affected by fluorescent proteins and antibodies. This labeling method allowed us to study T-cadherin clustering dynamics at the cell surface. HMW adiponectin induced the formation of stable T-cadherin clusters while LDL induced short-lived clusters. Cellular responses were also different: LDL triggered cholesterol- and actin-dependent calcium signaling without internalization while adiponectin promoted the opposite effect. CONCLUSIONS: We revealed distinct ligand-specific T-cadherin clustering and its ability to induce internalization or intracellular calcium signaling that likely explains the different physiological effects of LDL and HMW adiponectin. GENERAL SIGNIFICANCE: This work highlights the importance of GPI-anchored receptor clustering dynamics in mediating cellular responses. Different ligands can induce different effects in an identical cell via the same receptor.
BACKGROUND: Unlike other cadherins, T-cadherin does not mediate strong cell-cell adhesion. It has two soluble ligands: low density lipoprotein (LDL) and high-molecular-weight (HMW) adiponectin. LDL binding to T-cadherin induces calcium signaling, migration, and proliferation, and has proatherogenic effects, but adiponectin binding promotes antiatherogenic effects. The reasons for this difference and mechanism of signal transduction by glycosylphosphatidylinositol (GPI)-anchored T-cadherin are unknown. METHODS: We compared the ability of LDL and HMW adiponectin to induce calcium signaling, T-cadherin clustering and internalization. We measured calcium signaling in smooth muscle cells and T-cadherin expressing HEK293 using single-cell imaging. To study receptor clustering, we tested three different T-cadherin labeling strategies and then utilized confocal microscopy and flow cytometry assays based on Förster resonance energy transfer (FRET). RESULTS: Enzymatically labeled T-cadherin retained its cellular localization and physiological activity, features that were otherwise affected by fluorescent proteins and antibodies. This labeling method allowed us to study T-cadherin clustering dynamics at the cell surface. HMW adiponectin induced the formation of stable T-cadherin clusters while LDL induced short-lived clusters. Cellular responses were also different: LDL triggered cholesterol- and actin-dependent calcium signaling without internalization while adiponectin promoted the opposite effect. CONCLUSIONS: We revealed distinct ligand-specific T-cadherin clustering and its ability to induce internalization or intracellular calcium signaling that likely explains the different physiological effects of LDL and HMW adiponectin. GENERAL SIGNIFICANCE: This work highlights the importance of GPI-anchored receptor clustering dynamics in mediating cellular responses. Different ligands can induce different effects in an identical cell via the same receptor.