A three-amino-acid-long HLA-DRβ cytoplasmic tail is sufficient to overcome ER retention of invariant-chain p35

The cytoplasmic C-terminal domains of NR2 subunits have been proposed to modulate the assembly and trafficking of NMDA receptors. However, questions remain concerning which domains in the C terminus of NR2 subunits control the assembly of receptor complexes and how the assembled complexes are selectively trafficked through the various cellular compartments such as endoplasmic reticulum (ER) to the cell surface. In the present study, we found that the three amino acid tail after the TM4 region of NR2 subunits is necessary for surface expression of functional NMDA receptors, while truncations with only two amino acids following the TM4 region (NR2⌬2) completely eliminated surface expression of the NMDA receptor on co-expression with NR1-1a in HEK293 cells. FRET (fluorescence resonance energy transfer) analysis showed that these NR2⌬2 truncations are able to form homomers and heteromers on co-expression with NR1-1a. Furthermore, when NR2⌬2 subunits were cotransfected with either the NR1-4a or NR1-1a AAA mutant, lacking the ER retention motif (RRR), functional NMDA receptors were detected in the transfected HEK293 cells. Unexpectedly, we found that the replacement of five residues after TM4 with alanines gave results indistinguishable from those of NR2B⌬5 (EHLFY), demonstrating the short tail following the TM4 of NR2 subunits is not sequence-specific-dependent. Taken together, our results show that the C terminus of the NR2 subunits is not necessary for the assembly of NMDA receptor complexes, whereas a three amino acid long cytoplasmic tail following the TM4 of NR2 subunits is sufficient to overcome the ER retention existing in the C terminus of NR1, allowing the assembled NMDA receptors to reach the cell surface. N-methyl-D-aspartate (NMDA)3 receptors are heteromeric complexes primarily assembled from two subunit classes: NR1 and NR2. Co-assembly of NR1 and NR2 subunits is essential for formation of a functional channel, presumed to be a tetramer containing two NR1 and two NR2 subunits (1-2). NR1 is a single subunit with eight splicing variants, which have distinct trafficking and functional properties (3). NR2 subunits are coded by four separate genes, NR2A-D, each of which can endow the receptor channel with different properties (4). Thus, the subunit composition of NMDA receptors is a major determinant of NMDA receptor-mediated activity in the central nervous system. Although much is known about the physiological roles that NMDARs play in long term potentiation (LTP), learning and memory (5-7), much remains to be learned about the mechanisms by which these receptors are assembled, sorted, targeted, and anchored to the appropriate location.The C-terminal domains of the receptor subunits contain critical determinants of subcellular receptor localization. Regulation of receptor trafficking by these determinants ensures that only fully assembled multimeric receptors are expressed on the plasma membrane. When expressed alone in heterogeneous cells, the major NR1 isoform (NR1-1) is retained in the ER because of an RR...

Section: Discussionsupporting

confidence: 68%

A Three Amino Acid Tail Following the TM4 Region of the N-Methyl-D-aspartate Receptor (NR) 2 Subunits Is Sufficient to Overcome Endoplasmic Reticulum Retention of NR1-1a Subunit

Yang

Zheng

Song

et al. 2007

“…The ER retrieval activity of the RXR signal was decreased when it was positioned closer to the TM segment, whereas the opposite effect was observed for the KKXX signal. The loss of RXR signal activity in membrane proximity has also been supported by other studies using different membrane proteins (35,36). This may be related to the differential binding property of the RXR and KKXX motifs to the COPI proteins, as has been revealed by structural studies (37).…”

Section: Discussionsupporting

confidence: 55%

Role of C-terminal Membrane-proximal Basic Residues in Cell Surface Trafficking of HIV Coreceptor GPR15 Protein

Okamoto

Bernstein

Shikano

2013

“…The RXR-type ER localization signals have been reported in an increasing number of surface membrane proteins (21,25,29,30,33,(47)(48)(49). In many cases the RXR motif is thought to be exposed upon incomplete folding and/or assembly, hence serving as a checkpoint for protein quality control in the early secretory pathway (15,25,33,50).…”

Section: Discussionmentioning

confidence: 99%

“…Because a stretch of Arg residues (RXR motif) is known to mediate Golgi-to-ER retrograde transport of various membrane proteins (8,15,(25)(26)(27)(28)(29)(30), we tested if the C-terminal Argcontaining sequence of GPR15 possesses the ER localization activity. Truncation of the last two residues (Ser 359 and Leu 360 ) substantially reduced the surface expression (Fig.…”

Section: Gpr15 C Terminus Contains Er Localization Signal Thatmentioning

confidence: 99%

Phosphorylation-dependent C-terminal Binding of 14-3-3 Proteins Promotes Cell Surface Expression of HIV Co-receptor GPR15

Okamoto

Shikano

2011

Membrane trafficking is dictated by dynamic molecular interactions involving discrete determinants in the cargo proteins and the intracellular transport machineries. We have previously reported that cell surface expression of GPR15, a G protein-coupled receptor (GPCR) that serves as a co-receptor for HIV, is correlated with the mode III binding of 14-3-3 proteins to the receptor C terminus. Here we provide a mechanistic basis for the role of 14-3-3 in promoting the cell surface expression of GPR15. The Ala mutation of penultimate phospho-Ser (S359A) that abolishes 14-3-3 binding resulted in substantially reduced O-glycosylation and the cell surface expression of GPR15. The surface membrane protein CD8 fused with the C-terminal tail of GPR15 S359A mutant was re-localized in the endoplasmic reticulum (ER). In the context of S359A mutation, the additional mutations in the upstream stretch of basic residues (RXR motif) restored O-glycosylation and the cell surface expression. The RXR motif was responsible for the interaction with coatomer protein I (COPI), which was inversely correlated with the 14-3-3 binding and cell surface expression. These results suggest that 14-3-3 binding promotes cell surface expression of GPR15 by releasing the receptor from ER retrieval/retention pathway that is mediated by the interaction of RXR motif and COPI. Moreover, 14-3-3 binding substantially increased the stability of GPR15 protein. Thus 14-3-3 proteins play multiple roles in biogenesis and trafficking of an HIV co-receptor GPR15 to control its cell surface density in response to the phosphorylation signal.