Ampar Endocytosis In

Structurally, AMPARs are presumed to be heteropentameric structures assembled by combining homologous subunits. The current topology model suggests that each AMPAR subunit (GluR) consists of three transmembrane regions, MD1, MD3, and MD4, (MD, membrane domain), and a re-entrant membrane segment corresponding to MD2. The major intracellular carboxyl-terminal (CT) region contains potential sites for protein phosphorylation and binding motifs for GluR-interacting proteins, including adaptor proteins involved in forming clathrin-coated endocytic vesicles. Based on the mechanisms involved and functional consequences, clathrin-dependent AMPAR endocytosis can be classified into two distinct types: constitutive and regulated (stimulated)28,29. Constitutive endocytosis occurs across all AMPAR subunits and requires a seven amino acid stretch (EFCYKSR) in the most juxtamembrane region of the CT that is 100% conserved among all AMPAR GluR subunits30. Interfering with the constitutive pathway does not lead to detectable alterations in the number of

AMPARs on the cell surface, likely because of the existence of a constitutive AMPAR exocytosis pathway that counteracts the constitutive endocytosis29.

The regulated endocytosis, on the other hand, is specifically associated with AMPARs containing the GluR2 subunit28. This GluR2 specificity is mediated by several sequence motifs uniquely present within the CT region of the subunit, including a middle domain that binds to both NSF and AP2 clathrin adaptor31 and a terminal PDZ-binding domain that interacts with a number of PDZ-containing proteins such as GRIP and PICK132. In addition, systematic mutational analysis has recently identified a novel tyrosine phosphorylation-dependent endocytic motif (869YKEGYNVYG877) in the GluR2 CT that is absolutely required for insulin-stimulated AMPAR endocytosis (Figure 32.1)30.

Ampar Glur2

Figure 32.1. Schematic Diagram Shows the Amino-Acid Sequence in Carboxyl Terminal (CT) of AMPAR GluR2 Subunit. Sequences conserved among GluR1-4 (conserved) and those involved in its interaction with Y-ethylmaleimide Sensitive Factor (NSF) or PSD-95/Discs Large/Zona Occludens-1 domain-containing proteins (PDZ) are highlighted. Note that insulin-induced AMPAR endocytosis can be disrupted only in cells expressing mutants with the three tyrosine residues near the end of the GluR2 CT deleted. The amino acid sequence that covers these three tyrosine residues was used to construct the interference GluR23Y peptide.

Figure 32.1. Schematic Diagram Shows the Amino-Acid Sequence in Carboxyl Terminal (CT) of AMPAR GluR2 Subunit. Sequences conserved among GluR1-4 (conserved) and those involved in its interaction with Y-ethylmaleimide Sensitive Factor (NSF) or PSD-95/Discs Large/Zona Occludens-1 domain-containing proteins (PDZ) are highlighted. Note that insulin-induced AMPAR endocytosis can be disrupted only in cells expressing mutants with the three tyrosine residues near the end of the GluR2 CT deleted. The amino acid sequence that covers these three tyrosine residues was used to construct the interference GluR23Y peptide.

Though the detailed underlying mechanism remains unclear, a recent study has reported that insulin stimulates a2-adrenergic receptor endocytosis by phosphorylating one or more of three tyrosine residues in its CT region, thereby creating a binding site for the Grb2 adaptor protein33. By analogy, tyrosine phosphorylation of YKEGYNVYG may create a binding motif for a clathrin-coated adaptor protein such as Grb2. The increased interaction between phosphorylated AMPAR and clathrin-adaptor recruits the receptors to clathrin-coated pits allowing enhanced receptor endocytosis (Figure 32.2).

Figure 32.2. Proposed Model of Action for Tat-GluR23Y Interfering Peptide in Blocking AMPAR Endocytosis and LTD. (A) GluR2-dependent endocytosis of AMPARs is a common final step for LTD induction resulting from various stimuli such as low-frequency stimulation, insulin application, and mGluR1/5 activation. These LTD-producing stimuli, via different intracellular cascades, lead to activation of the tyrosine-containing endocytic signal (YKEGYNVYGIE) present in the GluR2 C-terminus (CT), and results in an interaction between the AMPAR and a clathrin-adaptor protein. Since it is the adaptors, and then clathrin, that are recruited to AMPARs, it is the GluR2 CT-adaptor interaction which initiates assembly of AMPAR-containing clathrin-coated vesicles. This leads to regulated endocytosis of the receptor, and hence, LTD. (B) Application of a membrane-permeable peptide containing this GluR2 tyrosine signal (Tat-YKEGYNVYGIE) competitively disrupts the GluR2 CT-adaptor interaction preventing AMPAR endocytosis and LTD. Because the Tat-GluR23Y peptide disrupts the adaptor interaction in a strict sequence-specific manner without affecting other steps of the signaling cascade, it should be a very specific LTD blocker. This peptide may be used to functionally repair synaptic dysfunctions caused by enhanced LTD such as drug addiction-related behavioral sensitization.

Figure 32.2. Proposed Model of Action for Tat-GluR23Y Interfering Peptide in Blocking AMPAR Endocytosis and LTD. (A) GluR2-dependent endocytosis of AMPARs is a common final step for LTD induction resulting from various stimuli such as low-frequency stimulation, insulin application, and mGluR1/5 activation. These LTD-producing stimuli, via different intracellular cascades, lead to activation of the tyrosine-containing endocytic signal (YKEGYNVYGIE) present in the GluR2 C-terminus (CT), and results in an interaction between the AMPAR and a clathrin-adaptor protein. Since it is the adaptors, and then clathrin, that are recruited to AMPARs, it is the GluR2 CT-adaptor interaction which initiates assembly of AMPAR-containing clathrin-coated vesicles. This leads to regulated endocytosis of the receptor, and hence, LTD. (B) Application of a membrane-permeable peptide containing this GluR2 tyrosine signal (Tat-YKEGYNVYGIE) competitively disrupts the GluR2 CT-adaptor interaction preventing AMPAR endocytosis and LTD. Because the Tat-GluR23Y peptide disrupts the adaptor interaction in a strict sequence-specific manner without affecting other steps of the signaling cascade, it should be a very specific LTD blocker. This peptide may be used to functionally repair synaptic dysfunctions caused by enhanced LTD such as drug addiction-related behavioral sensitization.

Functionally, unlike constitutive endocytosis, stimulating regulated AMPAR endocytosis leads to a rapid and persistent reduction in the number of AMPARs expressed on the postsynaptic membrane surface, and hence plays an important role in controlling synaptic strength28. The ability to reduce cell-surface AMPARs makes regulated endocytosis an ideal candidate mechanism for the expression of certain forms of LTD. Consistent with this conjecture, interfering with either of these GluR2 CT endocytic motifs by postsynaptic application of short "interference peptides" containing these endocytic motif sequences, tyrosine-containing sequences30, AP231, and PICK132, can prevent the expression of hippocampal CA1 homosynaptic LTD in brain slices. Moreover, although the facilitated endocytosis of postsynaptic AMPARs is principally observed in conjunction with hippocampal CA1 LTD, it appears to be a common mechanism involved in the expression of certain forms of LTD in many other areas of the brain, including the cerebellum4 and the NAc24. These studies have firmly established a critical role for AMPAR endocytosis in LTD expression. In addition, these results also suggest that these three GluR2 CT endocytic signals are indispensably involved in regulated AMPAR endocytosis and hence LTD expression, and that peptides interfering with the function of these signals may be used as specific inhibitors to probe the functional roles of LTD30.

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