N-glycosylation site tagging suggests a three transmembrane domain topology for the glutamate receptor GluR1

Zinc is essential for many cellular processes, and its concentration in the cell must be tightly controlled. The Zrt/IRT-like protein (ZIP) family of zinc transporters have recently been identified as the main regulators of zinc influx into the cytoplasm (1); however, little is known about their in vivo roles. Previously, we have shown that fear of intimacy (foi) encodes a putative member of the ZIP family that is essential for development in Drosophila (2). Here we demonstrate that FOI can act as an ion transporter in both yeast and mammalian cell assays and is specific for zinc. We also provide insight into the mechanism of action of the ZIP family through membrane topology and structure-function analyses of FOI. Our work demonstrates that Drosophila FOI is closely related to mammalian ZIP proteins at the functional level and that Drosophila represents an ideal system for understanding the in vivo roles of this family. In addition, this work indicates that the control of zinc by ZIP transporters may play a critical role in regulating developmental processes.

Section: Discussionmentioning

confidence: 99%

Drosophila fear of intimacy Encodes a Zrt/IRT-like Protein (ZIP) Family Zinc Transporter Functionally Related to Mammalian ZIP Proteins

Mathews

Wang

Eide

et al. 2005

“…Speculation of alternate glutamate receptor topology was suggested by several early investigations (15)(16)(17)(18)(19). Several subsequent systematic studies concluded that non-NMDA glutamate receptor subunits possessed a three-transmembrane domain architecture (11,12,20). By generating functional N-linked glycosylation mutants of the NMDA NR1 subunit, we tested the hypothesized existence of a conserved hairpin pore structure similar to that found in voltage-gated K ϩ channels (13), thus supporting the three-transmembrane domain model.…”

mentioning

confidence: 80%

An Alanine Residue in the M3-M4 Linker Lines the Glycine Binding Pocket of the N-Methyl-D-aspartate Receptor

Wood¹,

VanDongen

1997

While attempting to map a central region in the M3-M4 linker of the N-methyl-D-aspartate receptor NR1 subunit, we found that mutation of a single position, Ala-714, greatly reduced the apparent affinity for glycine. Proximal N-glycosylation localized this region to the extracellular space. Glycine affinities of additional Ala-714 mutations correlated with side chain volume. Substitution of alanine 714 with cysteine did not alter glycine sensitivity, although this mutant was rapidly inhibited by dithionitrobenzoate. Glycine protected the A714C mutant from modification by dithionitrobenzoate, whereas the co-agonist L-glutamate was ineffective. These experiments place Ala-714 in the glycine binding pocket of the N-methyl-D-aspartate receptor, a determination not predicted by previous structural models based on bacterial periplasmic binding protein homology.The N-methyl-D-aspartate (NMDA) 1 receptor is distinguished among the ionotropic glutamate receptors by several properties. The receptor possesses a unique Ca 2ϩ permeability and a voltage-dependent Mg 2ϩ block (1). These two features define the NMDA receptor's role in several important physiologic phenomena, including long term potentiation, neuronal development, and excitotoxicity (2). However, the functional characteristic that may most differentiate the NMDA receptor from other glutamate receptors is a requirement for the coagonist glycine (3, 4). Occupancy of both glutamate and glycine binding sites is necessary for receptor activation, and the two sites are allosterically coupled (5, 6). The distinct glycine binding site has been suggested to play a role in regulation of NMDA receptor desensitization (7). While several residues in the amino terminus of NR1 have been demonstrated to greatly influence glycine affinity, binding is also modulated by the contributing NR2 subunits (8). Indeed, the glycine binding site is a current experimental target for therapeutic intervention in the hours immediately following ischemic stroke (9).Despite functional differences among the glutamate receptors, several recent studies have converged upon a single topology model for the ionotropic glutamate receptor family (10 -14). Speculation of alternate glutamate receptor topology was suggested by several early investigations (15)(16)(17)(18)(19). Several subsequent systematic studies concluded that non-NMDA glutamate receptor subunits possessed a three-transmembrane domain architecture (11,12,20). By generating functional N-linked glycosylation mutants of the NMDA NR1 subunit, we tested the hypothesized existence of a conserved hairpin pore structure similar to that found in voltage-gated K ϩ channels (13), thus supporting the three-transmembrane domain model. An important consequence of the new three-transmembrane domain configuration is an extracellular location of the M3-M4 linker.Inconsistent with the conclusion from these topology studies, however, were findings from several groups that residues in the M3-M4 linker of non-NMDA glutamate receptors could be phosphorylated (2...

“…These are found at virtually all excitatory synaptic connections in the mammalian brain and mediate the majority of excitatory synaptic transmission (2,3). They are thought to be assembled as heteromers of most likely four (4 -6) or perhaps five (7,8) individual subunits that share similar membrane topology (9,10). Starting from the N-terminus this includes an extracellular N-terminal domain (NTD) of ϳ400 amino acids and an S1 domain that precedes the first transmembrane domain and is necessary for ligand binding.…”

mentioning

confidence: 99%

A Novel Anterograde Trafficking Signal Present in the N-terminal Extracellular Domain of Ionotropic Glutamate Receptors

Xia

Zastrow

Malenka

2002

Trafficking of ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors to and from the postsynaptic membrane plays an important role in regulating transmission at excitatory synapses. AMPA receptor subunits contain a large extracellular N-terminal domain that is important for receptor assembly (1). To further investigate the determinants of receptor assembly and surface expression, we have epitope-tagged the N-terminal domain of the AMPA receptor subunit, GluR1, and expressed it in human embryonic kidney 293 cells and hippocampal neurons. Full-length GluR1 was readily detected on the cell surface in both cell types. However, surface expression was profoundly decreased by deletion or replacement of nine amino acids in the extreme N terminus. Immunoprecipitation experiments demonstrated that the mutant GluR1 in which this sequence was deleted still interacts with GluR2, suggesting that mutant GluR1 is capable of at least partial assembly into heteromeric structures. The mutant forms of GluR1 co-localize with an endoplasmic reticulum marker suggesting that they are retained in this structure. These results suggest a specific function of a short sequence present in the N-terminal domain in controlling anterograde trafficking of ionotropic glutamate receptors.Ionotropic glutamate receptors are routinely subdivided into three classes termed ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) 1 receptors, N-methyl-D-aspartate receptors, and kainate receptors. These are found at virtually all excitatory synaptic connections in the mammalian brain and mediate the majority of excitatory synaptic transmission (2, 3). They are thought to be assembled as heteromers of most likely four (4 -6) or perhaps five (7, 8) individual subunits that share similar membrane topology (9, 10). Starting from the N-terminus this includes an extracellular N-terminal domain (NTD) of ϳ400 amino acids and an S1 domain that precedes the first transmembrane domain and is necessary for ligand binding. A reentrant loop follows this transmembrane domain and forms the pore for the channel. This is followed by a second transmembrane domain and a large extracellular loop that contains the S2 region of the ligand-binding domain. Finally, the protein transverses the membrane a third time and ends intracellularly with a 50 -100-amino acid C-terminal domain.A major means by which glutamatergic signaling is modulated in vivo is by membrane trafficking processes controlling the surface expression of receptors (11). Protein interactions with the C-terminal cytoplasmic domains of glutamate receptor subunits are thought to play important roles in regulating both anterograde trafficking to and endocytic removal from the neuronal plasma membrane (11). Extracellular surfaces of ionotropic glutamate receptors are well known to mediate ligand binding and to contribute to intersubunit interactions involved in the assembly of functional receptors (1). There is accumulating evidence that extracellular surfaces of glutamate receptors may also play a...