Article Figures & Data
Figures
- Figure 1.
KA2 subunits form homomultimeric receptors in heterologous cells. A, Schematic diagrams of the KA2 constructs used for the experiments. HA and FLAG tags are inserted after the last residue of KA2, and M1-M4 indicate the transmembrane domains. NH2, N terminus. B, KA2-HA and KA2-FLAG form an oligomeric complex in HEK293 cells. HEK293 cells were cotransfected with KA2-HA and KA2-FLAG as indicated. Cell lysate was immunoprecipitated (IP w/) using anti-HA or anti-FLAG antibody, and the precipitates were immunoblotted with anti-FLAG or anti-HA antibody, respectively.
- Figure 6.
Elimination of the arginine-rich domain or di-leucine motif in KA2 does not alter receptor function. A, Representative current traces evoked by fast application of glutamate (10 mm, 100 msec; gray bar) to HEK 293 cells expressing homomeric KA2(R/A) and KA2(R/A+LL908-9VV) receptors. Despite robust surface expression, these homomeric receptors remain unresponsive to glutamate. B, Representative current traces evoked by fast application of glutamate to cells expressing homomeric GluR5(Q), GluR5/KA2, GluR5/KA2(R/A), and GluR5/KA2(R/A+LL908-9VV) receptors. Heteromeric GluR5/KA2 receptors desensitized significantly faster than homomeric GluR5 receptors. C, Mean peak amplitudes (left half) and mean desensitization rates (right half) of heteromeric GluR5/KA2, GluR5/KA2(R/A), and GluR5/KA2(R/A+LL908-9VV) receptors were not significantly different (n = 4, 13, and 8, respectively, for amplitudes; n = 4, 12, and 8, respectively, for desensitization rates). The desensitization τ was determined by fitting the current decay with a single exponential function. The holding potential in each case was -70 mV. Also note that both the wild-type KA2 and the mutants are tagged with N-terminal myc epitopes.
- Figure 7.
The ER retention/retrieval signal is sterically masked by heteromeric assembly. A, Both GluR6 and GluR6/1 chimeras can assemble with KA2 in heterologous cells. HEK293 cells were cotransfected with myc-KA2 and GFP-GluR6 (or GFP-GluR6/1 chimera) as indicated. The cell lysate was immunoprecipitated (IP w/) using anti-myc antibody, and the precipitates were immunoblotted with anti-myc or anti-GFP antibodies. B, Surface expression of a KA2 mutant with a disrupted ER retention signal and heteromerically assembled wild-type KA2 shown by FAC quantification. HEK293 cells were transfected with myc-KA2 [or myc-KA2(R/A) mutant] alone or together with other subunits (GluR6, GluR1, or GluR6/1) as indicated. Data represent means ± SEM of fluorescence intensities from 50,000 transfected cells stained for surface expression of KA2. *p < 0.05 relative to KA2 minus primary antibody control, Student's unpaired t test.
- Figure 2.
Intracellular retention of KA2 homomeric receptors. Surface and intracellular expression of myc-KA2 and GFP-GluR6 in COS-7 cells is shown. Live cells were labeled with anti-myc or anti-AFP antibodies (Surface), permeabilized, and labeled with anti-KA2 or anti-GluR6 antibodies (Intracellular). The constructs used for the staining are illustrated at the top; white bars correspond to KA2 sequences, and gray bars correspond to GluR6 sequences. Asterisks indicate the antibodies used for staining. Note that there is no surface staining for KA2.
- Figure 4.
The C terminus of KA2 contains a signal sufficient and necessary for intracellular retention. A, Surface and intracellular expression of GluR6 and KA2 chimeras with swapped C termini, GFP-GluR6/KA2 (left) and myc-KA2/GluR6 (middle). Also shown is the surface and intracellular expression of another KA2 chimera with an appended GluR6 C terminus (right). Live cells were labeled with anti-AFP or anti-myc antibodies (Surface), permeabilized, and labeled with anti-KA2 or anti-GluR6 antibodies (Intracellular). The constructs used for the staining are illustrated at the top; white bars correspond to KA2 sequences, and gray bars correspond to GluR6 sequences. Asterisks indicate the antibodies used. B, Surface expression of wild-type and chimeric GluR6 and KA2 receptors evaluated by biotinylation. Transfected live HEK293 cells were surface-biotinylated with sulfo-NHS-S-S-biotin, and the biotinylated (Bio) and intracellular (Int) fractions of the cell extract were purified and immunoblotted with anti-myc or anti-GFP antibodies. Myc-KA2 (left panel, third lane) and GFP-GluR6/KA2 (right panel, fourth lane) are not present in the biotinylated fractions, whereas myc-KA2/G6 (left panel, fourth lane) and GFP-GluR6 (right panel, third lane) are detected.
- Figure 3.
The KA2 homomeric receptors are retained in the ER. A, KA2 colocalizes with ER markers. In the left panel, COS-7 cells transfected with myc-KA2 were permeabilized and stained with antibodies against myc (red) and the ER marker calnexin (green); in the right panel, COS-7 cells were cotransfected with myc-KA2 (green) and DsRed-ER (red). Image overlays show extensive colocalization (yellow) in both stains. B, KA2 does not localize to Golgi or tans-Golgi network compartments. COS-7 or NRK cells were transfected with myc-KA2 and costained with the Golgi markers giantin (green), mannosidase II (red), and TGN-38 (red). Image overlays showed no colocalization (yellow) in any stain.
- Figure 5.
Identification of an arginine-rich motif and a di-leucine sequence that mediate intracellular retention of KA2. A, Schematic diagram illustrating the structures of KA2 and Tac. M1-M4 indicate the four membrane-spanning segments. The KA2 C terminus has 155 amino acids; the arginine-rich motif and two di-leucine sequences are in bold letters and underlined. The arrows indicate the points of truncation to generate deletional mutants, open arrow for Δ113 and solid arrow for Δ118. Tac has a single transmembrane domain with a long N-terminal region and a short C-terminal tail. COOH, C terminus; NH2, N terminus. B, Surface and intracellular expression of Tac and TacKA2 mutants in COS cells. Tac and Tac-KA2 mutants containing deletion or point mutations of the arginine-rich motif are expressed on the cell surface. C, Staining of the same constructs expressed in the hippocampal neurons shows patterns identical to those in COS cells. D, Flow cytometric quantification for the surface expression of Tac and TacKA2 mutants in HEK293 cells. Note that the signal for TacKA2(R/A) is only ∼20% of those for Tac and TacKA2Δ118, whereas additional mutation (LL908-909VV) leads to full restoration of the surface expression. Data represent means ± SEM of fluorescence intensities from 50,000 transfected cells stained for surface expression of Tac or TacKA2 chimeras. *p < 0.05 relative to Tac minus primary antibody control, Student's unpaired t test.
- Figure 8.
Protein phosphorylation and a KA2-interacting protein do not regulate the ER retention of KA2. A, Surface (top) and intracellular (bottom) expression of TacKA2 (T858S859-DD), a phosphorylation-mimicking mutant. B, Surface (top) and intracellular (bottom left) expression of KA2, as well as intracellular expression of cotransfected SAP90 (bottom right). Note that there is no surface staining for either of the receptors.
