Regulation of Group I Metabotropic Glutamate Receptor Trafficking and Signaling by the Caveolar/Lipid Raft Pathway

Caveolin-1 interacts with mGluR1 via motifs located in the intracellular loops of the receptor. A, Mutations introduced in the putative caveolin-1-binding motifs present in the first (i1) and third (i3) intracellular loops of mGluR1. Critical aromatic residues were substituted with alanine. B, C, Mutations introduced in putative caveolin-1-binding motifs reduce caveolin-1 binding to mGluR1. GST-Cav-1 and control GST were incubated with wild-type and mutant mGluR1 immobilized to IgG-Sepharose. The receptor complex was eluted and probed with anti-GST antibody. Bound GST-Cav-1 is normalized to input receptor; binding to mutants is expressed as percentage of binding to wild-type. Shown are means ± SEM (n = 5; *p < 0.05, **p < 0.01, ***p < 0.001).

Caveolin-1 regulates constitutive mGluR1 internalization in HEK293 cells. A, B, Internalization of biotin-labeled wild-type and mutant receptors at 37°C for indicated times. A, Representative immunoblots probed with anti-mGluR1a antibody. B, Summary data for immunoblots like those in A; the percentage internalized receptor is calculated by subtracting abundance of intracellular receptor at t₀ from that at a given time point and normalizing to abundance of surface receptor at steady state (Surf, 4°C) [n = 3 (mut-i1 and mut-i3), n = 6 (mut-i1/i3), and n = 5 (wild type); t test, *p < 0.05, **p < 0.01]. C, D, Constitutive internalization of mGluR1 in cells expressing mGluR1-myc in the absence or presence of caveolin-1. C, Representative images of surface mGluR1-myc at steady state (4°C) and after incubation at 37°C for 15 or 30 min. Shown are cells expressing mGluR1-myc in absence (top) or presence (bottom) of caveolin-1. Scale bars, 50 μm. D, Summary of data in C. Internalization is defined as ratio of surface fluorescence at 37°C versus that at 4°C (n = 3; ≥50 cells analyzed per time point in each experiment). E, F, Internalization of mutant receptors lacking intact caveolin-1 binding motifs (mut-i1/3) in absence or presence of caveolin-1. E, Representative images of mut-i1/i3-myc expressed at the surface of HEK293 cells in absence (top) or presence (bottom) of caveolin-1 at steady state (4°C) and after incubation at 37°C for 15 or 30 min. Scale bars, 50 mm. F, Summary of data in E (n = 3). G, H, Internalization of mGluR1-myc in HEK293 cells transfected with control (top) and cav-1 (bottom) siRNAs. G, Representative images of surface receptors at steady state (4°C) or after incubation at 37°C for 5 or 15 min. H, Summary of data in G. Internalization is defined as ratio of surface fluorescence at 37°C versus that at 4°C; n ≥ 92 cells from three experiments; **p < 0.01. All data are means ± SEM.

Caveolin-1 regulates mGluR1 surface expression in HEK293 cells. Surface expression at steady state of wild-type and mutant mGluR1 lacking caveolin-1 binding motifs (mut-i1/3) expressed in transfected HEK293 cells. A, Representative images of myc-tagged wild-type and mutant receptors expressed at the cell surface (4°C). Scale bar, 10 μm. B, Representative immunoblots of biotin-labeled (surface) and total receptors probed with anti-mGluR1a antibody.

mGluR1 internalizes via the caveolar/raft pathway in HEK293 cells. A,B, Constitutive mGluR1-myc internalization is partially inhibited by cholesterol depletion with mβCD. A, Representative images of surface mGluR1-myc at steady state (4°C) and after internalization for 15 and 30 min at 37°C. B, Quantification of internalization from images like those in A assessed as loss of surface fluorescence after incubation at 37°C versus surface fluorescence at 4°C. Shown are means ± SEM from a representative experiment with n ≥ 111 cells analyzed per time point. C, Representative images and quantification of internalized (15 min) mGluR1-myc present in early endosomes marked by EEA1. Shown are means ± SEM (n = 13). D, Representative images and quantification of internalized (15 min) mGluR1-myc in vesicles labeled by ChTxB. Shown are means ± SEM (n = 33). E, Representative images and quantification of internalized (30 min) mGluR1-myc in vesicles positive for GFP-Cav-1. Shown are means ± SEM (n = 22). Colocalization appears in yellow in merged images. The arrowheads point to double-labeled vesicles. Scale bars, 10 μm.

mGluR1 colocalizes with markers of the caveolar/raft pathway in neurons. A, B, mGluR1 undergoes constitutive internalization in neurons and localizes to vesicles labeled by ChTxB. A, Representative images and quantification of internalized (37°C; 30 min) mGluR1-myc present in early endosomes marked by EEA1. Shown are means ± SEM (n = 15). B, Representative images and quantification of internalized mGluR1-myc (37°C; 30 min) present in ChTxB-positive vesicles. Shown are means ± SEM (n = 19). C, mGluR1-myc expressed at the neuronal surface localizes to discrete clusters that overlap with GFP-Cav1. Surface receptors were visualized by live labeling of neurons with anti-myc antibody (8°C; 30 min). Shown are means ± SEM (n = 14). D, GluR2 expressed at the neuronal surface of hippocampal neurons shows little overlap with caveolin-1. Shown are deconvolved epifluorescence images. Surface receptors were visualized by live labeling of neurons with an anti-GluR2 antibody (8°C; 30 min). Shown are means ± SEM (n = 49). The arrows point to areas of colocalization that appear in yellow in merged images; the boxed regions are shown magnified in insets. Scale bars, 10 μm.

Caveolin-1 regulates constitutive mGluR1 internalization and surface expression in neurons. A, Disruption of caveolin-1 binding motifs in mGluR1 decreases receptor surface expression in neurons. A, Representative images of surface and total mGluR1 (top panels) and mut-i1/3 (bottom panels) in hippocampal neurons at steady state. The boxed regions are shown magnified below. Scale bars, 50 μm. B, C, Constitutive internalization of wild-type mGluR1-myc and mut-i1/3-myc expressed in hippocampal neurons (DIV14). B, Representative images of surface and total receptors at steady state (8°C) and after internalization at 37°C for 10 and 20 min. Scale bars, 50 μm. C, Quantification of images like those in B; receptors internalization was calculated from normalized surface expression (surface/total fluorescence) at 37°C compared with the respective level at steady state (8°C). Shown are means ± SEM [mGluR1 (n ≥ 15), mut-i1/3 (n ≥ 12); *p < 0.05].

Native mGluR5 colocalizes with caveolin-1, associates with lipid rafts, and internalizes via the caveolar/raft pathway. A, Endogenous mGluR5 and caveolin-1 colocalize in neurons. Shown are representative images of a labeled hippocampal neuron at DIV9. Areas of colocalization are indicated by arrows and appear in yellow in the merged image. The boxed regions are shown magnified below. Scale bars, 10 μm. B, mGluR1/5 associate with lipid rafts in the brain. Rat brain membranes were extracted with Triton X-100 and separated on sucrose density gradients. Thirteen fractions were collected starting from the top of the gradient; equal volumes from each fraction were analyzed by immunoblot for the indicated proteins. Exposure time was kept constant (1 min) for all immunoblots except mGluR5 (10 s); input, total extract. Binding of ChTxB to GM1 was assayed by dot blot. C, D, Constitutive mGluR5 internalization in neurons is mediated by the caveolar/raft pathway. C, Representative immunoblots probed with anti-mGluR5 illustrating intracellular accumulation of biotinylated mGluR5 in control and treated cortical neurons. D, Quantification of intracellular biotin-labeled mGluR5; receptor internalization is expressed as fold increase of the ratio of intracellular versus total receptor compared with t₀ (4°C). Shown are means ± SEM (control, n = 7; K⁺ depletion, n = 7; nystatin, n = 5; *p < 0.05).