Tyrphostins: inhibitors of membrane traffic pathways?

Abstract

Several intracellular membrane trafficking events are mediated by tyrosine-containing motifs within the cytosolic domains of integral membrane proteins. Many such motifs conform to the consensus YXX+(+ represents a bulky hydrophobic residue). This motif interacts with the medium chain (mu) subunits of adaptor complexes which link the cytosolic domain of the integral membrane protein to the clathrin coat involved in vesicle formation. The motif YXX+ is similar to motifs which are phosphorylated by tyrosine kinases. Tyrphostins (structural analogues of tyrosine) are inhibitors of tyrosine kinases: can they inhibit interactions between mu chains and YXX+ motifs? We have investigated several tyrphostins and found that, in vitro and in the yeast two hybrid system, some are capable of inhibiting the intraction between YXX+ motifs and different medium chains. We have also shown that tyrphostins which block these interactions affect the internalisation and recyling of the transferrin receptor in live cells.At present little is known regarding the mechanism of metabotopic glutamate receptor (mGluR) trafficking. In order to study this, we inserted a hemagglutinin (HA) epitope tag between residues 57-58 of the extracellular domain of the rat mGluRla splice variant. In HEK293 cells transiently transfected with HA-mGlula, the epitope tagged receptor was primarily localized to the cell surface prior to agonist stimulation. Following stimulation with glutamate (1OpM; 30 min) the HA-mGluRla internalized to early endosomes and an endocytic recycling compartment. Further quantification of receptor endocytosis was provided by ELISA experiments which showed that the HA-mGluRla underwent rapid agonist-(glutamate 10 inpM) induced internalization with a t o,5 of 19 ? 3 min. To determine whether agonist-induced mGluRla internalization is an arrestin-and dynamin-dependent process cells were cotransfected with HA-mGluRla and either dynamin-K44A or arrestin-2 (319-418). Expression of either dominant negative mutant construct with HA-mGluRla strongly inhibited glutmate-induced (10 pM; 30 min) internalization. In addition arrestin-2-GFP or arrestin-3-GFP underwent agonist-induced translocation from cytosol to membrane in HEK293 cells coexpressing HA-mGluRla. Taken together our observations demonstrate that agonist-induced internalization of mGluRla is an arrestin-and dynamin-dependent process.Arrestin-and Dynamin-dependent. 73The effect of pertussis toxin on the internalisation of the Somatostatin agonists are rapidly and efficiently endocytosed with the somatostatin2 receptor. The aim of this study was to determine the role of G protein coupling on internalisation by examining the effect of pertussis toxin treatment. The accumulation of internalised agonists and receptors is governed by a number of processes including hormone-receptor association, dissociation, receptor endocytosis and recycling back to the plasma membrane. We have established a mathematical model describing these processes and used this model in the mechanistic interpretat...

Single-Molecule Analysis of PIP2;1 Dynamics and Partitioning Reveals Multiple Modes of Arabidopsis Plasma Membrane Aquaporin Regulation

“…TyrA23 can specifically prevent the interaction between cargo motifs destined for endocytosis and the m2 subunit of the clathrin binding AP-2 adaptor complex (Banbury et al, 2003). A recent report showed that TyrA23 efficiently inhibited interaction between the human transferrin receptor and a m-adaptin subunit and then blocked the internalization of the human transferrin receptor in Arabidopsis protoplasts (Ortiz-Zapater et al, 2006).…”

“…TyrA23 is thought to block specifically the interaction of endocytic cargo motifs with the clathrin medium chain. This interaction is crucial for establishing a link between the cytosolic domains of membrane proteins to the clathrin coat involved in vesicle formation (Banbury et al, 2003). Exposure of seedlings to TyrA23 led to an overall decrease in GFP-PIP2;1 diffusion coefficients, with Ĝ reduced to 1.59 3 10 23 mm 2 /s (SE 1.41 to 1.78 3 10 23 mm 2 /s) ( Figure 9A; see Supplemental Movie 6 online).…”

“…Also, the effects of TyrA23 were found to be reversible; at 20 min after washout, the Ĝ value had increased to 2.20 3 10 23 mm 2 /s (SE 1.88 to 2.58 3 10 23 mm 2 /s) (see Supplemental Figure 8A online), comparable to that of the control cells. When tyrphostin A51, an analog of TyrA23 that does not interfere with the clathrin-cargo interaction, was used (Banbury et al, 2003;, the diffusion coefficient of GFP-PIP2;1 did not change (see Supplemental Figure 8B online). The recovery experiment and noneffective analog treatment clearly suggested that the concentrations of TyrA23 used in this study were within the physiological range and its effect is rather specific.…”

Identification and Dynamics of Arabidopsis Adaptor Protein-2 Complex and Its Involvement in Floral Organ Development

“…Mutations of the YXXF motif abolish the interaction with m (Ohno et al, 1995;Boll et al, 1996;Stephens et al, 1997) and alter the subcellular localization of the cargo proteins (Bos et al, 1993;Humphrey et al, 1993). The interaction of m with cargo proteins through the YXXF motif is disrupted by the structural Tyr analog Tyrphostin A23 (TyrA23), which is an efficient inhibitor of clathrin-mediated endocytosis (Banbury et al, 2003). Evidence for the importance of AP-2 during development is suggested by studies of mutants with disrupted subunit genes.…”

“…To examine whether the plasma membrane localization of AP2M-GFP is dependent on the interaction with cargo proteins, the root tip cells of the AP2M-GFP ap2m seedlings were incubated with the clathrinmediated endocytosis inhibitor TyrA23. Tyrphostin A51 (TyrA51), which has no effect on the interaction between AP-2 and cargo proteins (Banbury et al, 2003), was used as the negative control. TyrA23 significantly reduced the AP2M-GFP fluorescence at the plasma membrane in a time-dependent manner, although the fluorescence at the plasma membrane was not altered by TyrA51 even after the 2-h incubation ( Figure 1C).…”