argenx BVBA.

In renal collecting ducts, a vasopressin-induced cAMP increase results in the phosphorylation of aquaporin-2 (AQP2) water channels at Ser-256 and its redistribution from intracellular vesicles to the apical membrane. Hormones that activate protein kinase C (PKC) proteins counteract this process. To determine the role of the putative kinase sites in the trafficking and hormonal regulation of human AQP2, three putative casein kinase II (Ser-148, Ser-229, Thr-244), one PKC (Ser-231), and one protein kinase A (Ser-256) site were altered to mimic a constitutively non-phosphorylated/phosphorylated state and were expressed in Madin-Darby canine kidney cells. Except for Ser-256 mutants, seven correctly folded AQP2 kinase mutants trafficked as wild-type AQP2 to the apical membrane via forskolin-sensitive intracellular vesicles. With or without forskolin, AQP2-Ser-256A was localized in intracellular vesicles, whereas AQP2-S256D was localized in the apical membrane. Phorbol 12-myristate 13-acetate-induced PKC activation following forskolin treatment resulted in vesicular distribution of all AQP2 kinase mutants, while all were still phosphorylated at Ser-256. Our data indicate that in collecting duct cells, AQP2 trafficking to vasopressinsensitive vesicles is phosphorylation-independent, that phosphorylation of Ser-256 is necessary and sufficient for expression of AQP2 in the apical membrane, and that PMA-induced PKC-mediated endocytosis of AQP2 is independent of the AQP2 phosphorylation state.In humans, the kidney is the prime organ for regulation of body fluid osmolarity, which is maintained within strict boundaries. To fine-tune this balance, principal cells of the renal collecting duct reabsorb water from pro-urine, which is under control of the anti-diuretic hormone arginine vasopressin (AVP).1 Upon hypovolemia or hypernatremia, pituitary-derived AVP binds its V2 receptor in the basolateral membrane of these cells and initiates an intracellular cAMP signaling cascade that causes a transient increase in cytosolic calcium (1) and the activation of protein kinase A (PKA), which in turn phosphorylates homotetrameric aquaporin-2 (AQP2) water channels and possibly other proteins. Consequently, AQP2-containing vesicles fuse with the apical membrane, rendering the principal cells water-permeable (2, 3). Driven by an osmotic gradient, water will then enter these cells via AQP2 and will exit the cells via AQP3 and AQP4, located in the basolateral membrane, a process in which urine is concentrated. By using antibodies that recognize Ser-256-phosphorylated AQP2 (p-AQP2), Nishimoto et al. (4) were able to show that in vivo AVP-induced redistribution of AQP2 from vesicles to the apical membrane coincides with phosphorylation of Ser-256. By using similar antibodies, Christensen et al. (5) demonstrated that p-AQP2 is, besides the apical membrane, also present in intracellular vesicles of principal cells and that the intracellular distribution of AQP2 is regulated via V2 receptors by altering the phosphorylation state of Ser-256 in AQ...

Fluorescence-anisotropy-based homo-FRET detection methods can be employed to study clustering of identical proteins in cells. Here, the potential of fluorescence anisotropy microscopy for the quantitative imaging of protein clusters with subcellular resolution is investigated. Steady-state and time-resolved anisotropy detection and both one- and two-photon excitation methods are compared. The methods are evaluated on cells expressing green fluorescent protein (GFP) constructs that contain one or two FK506-binding proteins. This makes it possible to control dimerization and oligomerization of the constructs and yields the experimental relation between anisotropy and cluster size. The results show that, independent of the experimental method, the commonly made assumption of complete depolarization after a single energy transfer step is not valid here. This is due to a nonrandom relative orientation of the fluorescent proteins. Our experiments show that this relative orientation is restricted by interactions between the GFP barrels. We describe how the experimental relation between anisotropy and cluster size can be employed in quantitative cluster size imaging experiments of other GFP fusions. Experiments on glycosylphosphatidylinisotol (GPI)-anchored proteins reveal that GPI forms clusters with an average size of more than two subunits. For epidermal growth factor receptor (EGFR), we observe that approximately 40% of the unstimulated receptors are present in the plasma membrane as preexisting dimers. Both examples reveal subcellular heterogeneities in cluster size and distribution.

The suggestion that microdomains may function as signaling platforms arose from the presence of growth factor receptors, such as the EGFR, in biochemically isolated lipid raft fractions. To investigate the role of EGFR activation in the organization of lipid rafts we have performed FLIM analyses using putative lipid raft markers such as ganglioside GM1 and glycosylphosphatidylinositol (GPI)-anchored GFP (GPI-GFP). The EGFR was labeled using single domain antibodies from Llama glama that specifically bind the EGFR without stimulating its kinase activity. Our FLIM analyses demonstrate a cholesterol-independent colocalization of GM1 with EGFR, which was not observed for the transferrin receptor. By contrast, a cholesterol-dependent colocalization was observed for GM1 with GPI-GFP. In the resting state no colocalization was observed between EGFR and GPI-GFP, but stimulation of the cell with EGF resulted in the colocalization at the nanoscale level of EGFR and GPI-GFP. Moreover, EGF induced the enrichment of GPI-GFP in a detergent-free lipid raft fraction. Our results suggest that EGF induces the coalescence of the two types of GM1-containing microdomains that might lead to the formation of signaling platforms.

The current activation model of the EGF receptor (EGFR) predicts that binding of EGF results in dimerization and oligomerization of the EGFR, leading to the allosteric activation of the intracellular tyrosine kinase. Little is known about the regulatory mechanism of receptor oligomerization. In this study, we have employed FRET between identical fluorophores (homo-FRET) to monitor the dimerization and oligomerization state of the EGFR before and after receptor activation. Our data show that, in the absence of ligand, ϳ40% of the EGFR molecules were present as inactive dimers or predimers. The monomer/predimer ratio was not affected by deletion of the intracellular domain. Ligand binding induced the formation of receptor oligomers, which were found in both the plasma membrane and intracellular structures. Ligand-induced oligomerization required tyrosine kinase activity and nine different tyrosine kinase substrate residues. This indicates that the binding of signaling molecules to activated EGFRs results in EGFR oligomerization. Induction of EGFR predimers or preoligomers using the EGFR fused to the FK506-binding protein did not affect signaling but was found to enhance EGF-induced receptor internalization. Our data show that EGFR oligomerization is the result of EGFR signaling and enhances EGFR internalization.The EGF receptor (EGFR 2 ; ErbB1) has an essential role in the regulation of growth and differentiation of a large range of cell types. The EGFR belongs to the ErbB family, all four members of which have been implicated in the development of different cancers (1). The first step in the signal transduction cascade is the binding of its ligand such as EGF or TGF-␣ to the ectodomain, which provokes receptor dimerization and oligomerization. Deletion of the dimerization domain, which is present in domain II of the EGFR ectodomain, blocks receptor activation completely, demonstrating that receptor dimerization is critical for the allosteric activation of the tyrosine kinase (2, 3). Activation of the receptor tyrosine kinase results in cross-phosphorylation of the receptors, and the phosphotyrosines in the intracellular domain serve subsequently as docking sites for adaptor proteins such as Grb2 and Shc and enzymes such as phospholipase C␥, which contain phosphotyrosine-specific SH2 (Src homology 2) or phosphotyrosine-binding domains. Eventually, the active ligand-receptor complex becomes internalized via both clathrin-dependent and clathrin-independent pathways, followed by the intracellular transport to lysosomes, where the ligand-receptor complexes are degraded (4).Although EGF binding and dimerization seem to be strictly connected, both microscopic and biochemical studies have demonstrated that, in resting cells, the receptor is already found on the cell surface as non-active dimers, the so-called predimers. This phenomenon was initially discovered using electron microscopy and immunogold labeling of the EGFR: in the resting cell, ϳ35% of the total receptor population was present as receptor predimers (5). Th...