Interactions between intracellular domains as key determinants of the quaternary structure and function of receptor heteromers

Abstract

G protein-coupled receptor (GPCR) heteromers are macromolecular complexes with unique functional properties different from those of its individual protomers. Little is known about what determines the quaternary structure of GPCR heteromers resulting in their unique functional properties. In this study, using resonance energy transfer techniques in experiments with mutated receptors, we provide for the first time clear evidence for a key role of intracellular domains in the determination of the quaternary structure of GPCR heteromers between adenosine A(2A), cannabinoid CB(1), and dopamine D(2) receptors. In these interactions, arginine-rich epitopes form salt bridges with phosphorylated serine or threonine residues from CK1/2 consensus sites. Each receptor (A(2A), CB(1), and D(2)) was found to include two evolutionarily conserved intracellular domains to establish selective electrostatic interactions with intracellular domains of the other two receptors, indicating that these particular electrostatic interactions constitute a general mechanism for receptor heteromerization. Mutation experiments indicated that the interactions of the intracellular domains of the CB(1) receptor with A(2A) and D(2) receptors are fundamental for the correct formation of the quaternary structure needed for the function (MAPK signaling) of the A(2A)-CB(1)-D(2) receptor heteromers. Analysis of MAPK signaling in striatal slices of CB(1) receptor KO mice and wild-type littermates supported the existence of A(1)-CB(1)-D(2) receptor heteromer in the brain. These findings allowed us to propose the first molecular model of the quaternary structure of a receptor heteromultimer.

FIGURE 1.

A_2A-CB₁-D₂ receptor heteromerization in living cells. Assays were performed 48 h post-transfection in cells expressing A_2A-Rluc receptor (1 μg of cDNA; ∼100,000 luminescence units), D₂-GFP² receptor (3 μg of cDNA; ∼6,000 fluorescence units), and increasing amounts of CB₁-YFP receptor cDNA (8,000–18,000 fluorescence units). In each sample fluorescence or luminescence was measured before every experiment to confirm similar donor expressions while monitoring the increased acceptor expression. a and b, aliquots of these cells were used. a, net SRET² was obtained by monitoring the YFP fluorescence emission after DeepBlueC addition, with subtraction of the value obtained with cells expressing the same amount of A_2A-Rluc and D₂-GFP² receptors. SRET saturation curves (black) were obtained for the coupling of A_2A-Rluc, D₂-GFP², and CB₁-YFP receptors, although negligible and linear SRET was obtained in cells expressing equivalent amounts of A_2A-Rluc, D₂-GFP², and 5HT_2B-YFP receptors (green) or D₄-Rluc, A_2A-GFP², and CB₁-YFP receptors (red). SRET data are expressed as means ± S.D. of 5–8 different experiments grouped as a function of the amount of SRET acceptor. b, BRET¹ was obtained by monitoring the YFP fluorescence emission after coelenterazine H addition, with subtraction of the value obtained with cells expressing the same amount of A_2A-Rluc receptor. BRET² was obtained by monitoring the emission of GFP² fluorescence after DeepBlueC addition, with subtraction of the value obtained with cells expressing the same amount of A_2A-Rluc receptors. FRET was measured by monitoring the emission of YFP fluorescence after excitation of GFP² at 400 nm. Data are expressed as the mean ± S.E. of 5–8 independent experiments performed in duplicate. Linear unmixing of the emission signals was applied to BRET² and FRET values (b) and for YFP quantification in saturation curves (a). c, schematic representation of the putative triangular quaternary structure of the A_2A-CB₁-D₂ receptor heteromer. mBu, milli-BRET unit.

FIGURE 2.

A_2A-CB₁^A467-A468-D₂ receptor heteromerization in living cells. Assays were performed 48 h post-transfection in cells expressing the following: a, A_2A-Rluc receptor (1 μg of cDNA; ∼100,000 luminescence units) and increasing amounts of cDNA of the CB₁-YFP or CB₁^A467-A468-YFP receptors (8,000–18,000 fluorescence units); mBu, milli-BRET unit. b, D₂-GFP² (3 μg of the cDNA; ∼6,000 fluorescence units) and increasing amounts of the cDNA for CB₁-YFP or CB₁^A467-A468-YFP; c and d, A_2A-Rluc receptor (1 μg of cDNA; ∼100,000 luminescence units), D₂-GFP² receptor (3 μg of the cDNA; ∼6,000 fluorescence units), and increasing amounts of cDNA of the CB₁^A467-A468-YFP receptor (8,000–18,000 fluorescence units). In each sample, fluorescence or luminescence was measured before every experiment to confirm similar donor expressions while monitoring the increased acceptor expression. a, BRET¹ saturation curves for the A_2A-Rluc-CB₁-YFP receptor pair (squares) and for the A_2A-Rluc-CB₁^A467-A468-YFP receptor pair (triangles) were obtained by monitoring the YFP fluorescence emission after coelenterazine H addition, with subtraction of the value obtained with cells expressing the same amount of A_2A-Rluc receptor. Data are expressed as means ± S.D. of five different experiments grouped as a function of the amount of BRET¹ acceptor. b, FRET saturation curves for the D₂-GFP²-CB₁-YFP receptor pair (triangles) and for the D₂-GFP²- CB₁^A467-A468-YFP receptor pair (squares) were obtained by monitoring the YFP fluorescence emission at 530 nm after excitation of GFP² at 400 nm, with subtraction of the value obtained with cells expressing the same amount of donor protein. Data are expressed as means ± S.D. of seven different experiments grouped as a function of the amount of FRET acceptor. c, net SRET² was obtained by monitoring the emission of YFP fluorescence after DeepBlueC addition, with subtraction of the value obtained with cells expressing the same amount of A_2A-Rluc and D₂-GFP² receptors. SRET saturation curves (solid line) were obtained for the coupling of A_2A-Rluc, D₂-GFP², and CB₁^A467-A468-YFP receptors and compared with the curve obtained for the coupling of A_2A-Rluc, D₂-GFP², and CB₁-YFP receptors (dotted line, see Fig. 1). SRET data are expressed as means ± S.D. of five different experiments grouped as a function of the amount of SRET acceptor. d, BRET¹, BRET², and FRET were measured as indicated in Fig. 1 legend. Data are expressed as % of values obtained in cells expressing A_2A-Rluc, D₂-GFP², and CB₁-YFP receptors (control, Fig. 1b), in mean ± S.E. of five independent experiments performed in duplicate. One-way ANOVA followed by Bonferroni test showed significant increases or decreases with respect to the control (*, p < 0.05; **, p < 0.01; ***, p < 0.005). Linear unmixing of the emission signals was applied to the data for BRET² and FRET values (b and d) and for YFP quantification in saturation curves (a and c). e, the spectrum of a mixture of the following three peptides SVSTDAAAE, SVSTDpTpSAE, and LRIFLAARR, shows only one noncovalent complex between SVSTDpTpSAE and LRIFLAARR at 2171.7 atomic mass units (see text).

FIGURE 3.

A_2A^A205-A206-CB₁-D₂ receptor heteromerization in living cells. Assays were performed 48 h post-transfection in cells expressing the following: a, A_2A-Rluc or A_2A^A205-A206-Rluc receptors (1 or 0.8 μg of cDNA respectively; ∼100,000 luminescence units) and increasing amounts of the cDNA of the CB₁-YFP receptor (8,000–18,000 fluorescence units). mBu, milli-BRET unit. b, A_2A-Rluc or A_2A^A205-A206-Rluc (1 or 0.8 μg of cDNA, respectively; ∼100,000 luminescence units) and increasing amounts of the cDNA for D₂-YFP. c and d, A_2A^A205-A206-Rluc receptor (1 μg of cDNA; ∼100,000 luminescence units), D₂-GFP² receptor (3 μg of the cDNA; ∼6,000 fluorescence units), and increasing amounts of cDNA of the CB₁-YFP receptor (8,000–18,000 fluorescence units). In each sample fluorescence or luminescence was measured before every experiment to confirm similar donor expressions while monitoring the increased acceptor expression. a, BRET¹ saturation curves for the A_2A-Rluc-CB₁-YFP receptor pairs (squares) and for the A_2A^A205-A206-Rluc-CB₁-YFP receptor pair (triangles) were obtained by monitoring the YFP fluorescence emission after coelenterazine H addition, with subtraction of the value obtained with cells expressing the same amount of donor. Data are expressed as means ± S.D. of five different experiments grouped as a function of the amount of BRET¹ acceptor. b, BRET¹ saturation curves for the A_2A-Rluc-D₂-YFP receptor pairs (triangles) and for the A_2A^A205-A206-Rluc-D₂-YFP receptor pair (squares) were obtained by monitoring the YFP fluorescence emission after coelenterazine H addition, with subtraction of the value obtained with cells expressing the same amount of donor. Data are expressed as means ± S.D. of five different experiments grouped as a function of the amount of BRET¹ acceptor. c, net SRET² was obtained by monitoring the YFP fluorescence emission after DeepBlueC addition, with subtraction of the value obtained with cells expressing the same amount of A_2A^A205-A206-Rluc and D₂-GFP² receptors. SRET saturation curves (solid line) were obtained for the coupling of A_2A^A205-A206Rluc, D₂-GFP², and CB₁-YFP receptors and compared with the curve obtained for the coupling of A_2A-Rluc, D₂R-GFP², and CB₁-YFP receptors (dotted line, see Fig. 1). SRET data are expressed as means ± S.D. of five different experiments grouped as a function of the amount of SRET acceptor. d, BRET¹, BRET², and FRET were measured as indicated in Fig. 1 legend. Data are expressed as % of values obtained in cells expressing A_2A-Rluc, D₂-GFP², and CB₁-YFP receptors (control, Fig. 1b), in mean ± S.E. of five independent experiments performed in duplicate. One-way ANOVA followed by Bonferroni test showed significant increases or decreases with respect to the control (**, p < 0.01; ***, p < 0.005). Linear unmixing of the emission signals was applied to the data for BRET² and FRET values (d) and for YFP quantification in saturation curves (a–c). e, spectrum of a mixture of the following three peptides LRIFLAAAA, LRIFLAARR, and SVSTDpTpSAE, shows only one NCX between SVSTDpTpSAE and LRIFLAARR at 2171.7 atomic mass units (see text).

FIGURE 4.

A_2A-CB₁^A321-A322-D₂ receptor heteromerization in living cells. Assays were performed 48 h post-transfection in cells expressing the following: a, D₂-Rluc receptor (1 μg of cDNA; ∼100,000 luminescence units) and increasing amounts of the cDNA for CB₁-YFP or CB₁^A321-A322-YFP receptors (8,000–18,000 fluorescence units); b, A_2A-Rluc (1 μg of cDNA; ∼100,000 luminescence units) and increasing amounts of the cDNA for CB₁-YFP or CB₁^A321-A322-YFP; c and d, A_2A-Rluc receptor (1 μg of cDNA; ∼100,000 luminescence units), D₂-GFP² receptor (3 μg of the cDNA; ∼6,000 fluorescence units), and increasing amounts of cDNA of the CB₁^A321-A322-YFP receptor (8,000–18,000 fluorescence units). In each sample fluorescence or luminescence was measured before every experiment to confirm similar donor expressions while monitoring the increased acceptor expression. a, BRET¹ saturation curves for the D₂-Rluc-CB₁-YFP receptor pair (squares) and for D₂-Rluc-CB₁^A321-A322-YFP receptor pair (triangles) were obtained by monitoring the YFP fluorescence emission after coelenterazine H addition, with subtraction of the value obtained with cells expressing the same amount of A_2A-Rluc receptor. Data are expressed as means ± S.D. of six different experiments grouped as a function of the amount of BRET¹ acceptor. b, BRET¹ saturation curves for the A_2A-Rluc-CB₁-YFP receptor pair (triangles) and for A_2A-Rluc-CB₁^A321-A322-YFP receptor pair (squares) were obtained by monitoring the YFP fluorescence emission after coelenterazine H addition, with subtraction of the value obtained with cells expressing the same amount of A_2A-Rluc receptor. Data are expressed as means ± S.D. of six different experiments grouped as a function of the amount of BRET¹ acceptor. c, net SRET² was obtained by monitoring the YFP fluorescence emission after DeepBlueC addition, with subtraction of the value obtained with cells expressing the same amount of A_2A-Rluc and D₂-GFP² receptors. SRET saturation curves (solid line) were obtained for the coupling of A_2A-Rluc, D₂-GFP², and CB₁^A321-A322-YFP receptors and compared with the curve obtained for the coupling of A_2A-Rluc, D₂-GFP², and CB₁-YFP receptors (dotted line, see Fig. 1). SRET data are expressed as means ± S.D. of six different experiments grouped as a function of the amount of SRET acceptor. d, BRET¹, BRET², and FRET were measured as indicated in Fig. 1b legend. Data are expressed as percent of values obtained in cells expressing A_2A-Rluc, D₂-GFP², and CB₁-YFP receptors (control, Fig. 1b), in mean ± S.E. of six independent experiments performed in duplicate. One-way ANOVA followed by Bonferroni test showed significant increases or decreases with respect to the control (*, p < 0.05; **, p < 0.01). Linear unmixing of the emission signals was applied to the data for BRET² and FRET values (d) and for YFP quantification in saturation curves (a–c). e, the spectrum of a mixture of the following three peptides AAEDGKVQVT, pTpSEDGKVQVT, and VLRRRRKRVN shows only one NCX between pTpSEDGKVQVT and VLRRRRKRVN at 2575.6 atomic mass units (see text). mBu, milli-BRET unit.

FIGURE 5.

A_2A-CB₁-D_2S receptor heteromerization in living cells. Assays were performed 48 h post-transfection in cells expressing the following: a, D_2S-GFP² receptor (1.5 μg of cDNA; ∼5,000 fluorescence units) or D₂-GFP² receptor (2 μg of cDNA; ∼5,000 luminescence units), and increasing amounts of cDNA of CB₁-YFP receptor (8,000–18,000 fluorescence units); b, A_2A-Rluc (1 μg of cDNA; ∼100,000 luminescence units) and increasing amounts of cDNA for D₂-GFP² or D_2S-GFP²; c and d, A_2A-Rluc receptor (1 μg of cDNA; ∼100,000 luminescence units), D_2S-GFP² receptor (3 μg of the cDNA; ∼6,000 fluorescence units) and increasing amounts of the cDNA for CB₁-YFP receptor (8,000–18,000 fluorescence units). mBu, milli-BRET unit. In each sample fluorescence or luminescence was measured before every experiment to confirm similar donor expressions while monitoring the increased acceptor expression. a, FRET saturation curves for the D₂-GFP²-CB₁-YFP receptor pair (squares) and for D_2S-GFP²-CB₁-YFP receptor pair (triangles) were obtained by monitoring the YFP fluorescence emission at 530 nm after excitation of GFP² at 400 nm, with subtraction of the value obtained with cells expressing the same amount of donor protein. Data are expressed as means ± S.D. of seven different experiments grouped as a function of the amount of FRET acceptor. b, BRET² saturation curves for the A_2A-Rluc-D₂-GFP² receptor pair (triangles) and for A_2A-Rluc-D_2S-GFP² receptor pair (squares) were obtained by monitoring the YFP fluorescence emission after DeepBlueC addition, with subtraction of the value obtained with cells expressing the same amount of A_2A-Rluc receptor. Data are expressed as means ± S.D. of six different experiments grouped as a function of the amount of BRET² acceptor. c, net SRET² was obtained by monitoring the YFP fluorescence emission after DeepBlueC addition, with subtraction of the value obtained with cells expressing the same amount of A_2A-Rluc and D_2S-GFP² receptors. SRET saturation curves (solid line) were obtained for the coupling of A_2A-Rluc, D_2S-GFP², and CB₁-YFP receptors and compared with the curve obtained for the coupling of A_2A-Rluc, D₂-GFP², and CB₁-YFP receptors (dotted line, see Fig. 1). SRET data are expressed as means ± S.D. of five different experiments grouped as a function of the amount of SRET acceptor. d, BRET¹, BRET², and FRET were measured as indicated in Fig. 1 legend. Data are expressed as % of values obtained in cells expressing A_2ARluc, D₂-GFP², and CB₁-YFP receptors (control, Fig. 1b), in mean ± S.E. of five independent experiments performed in duplicate. One-way ANOVA followed by Bonferroni test showed significant increases or decreases with respect to the control (***, p < 0.005). Linear unmixing of the emission signals was applied to the data for BRET² and FRET values (a, b, and d) and for YFP quantification in saturation curves (a and c). e, spectrum of a mixture of the following three peptides AAEDGKVQVT, pTpSEDGKVQVT, and NRRRVEAARR, shows only one NCX between pTpSEDGKVQVT and NRRRVEAARR at 2506.8 atomic mass units (see text).

FIGURE 6.

A_2A^A374-CB₁-D₂ receptor heteromerization in living cells. Assays were performed 48 h post-transfection in cells expressing the following: a, A_2A-Rluc or A_2A^A374-Rluc receptors (1 or 0.8 μg of cDNA respectively; ∼100,000 luminescence units) and increasing amounts of cDNA of the D₂-YFP receptor (8,000–18,000 fluorescence units); b, A_2A-Rluc or A_2A^A374-Rluc (1 or 0.8 μg of cDNA respectively; ∼100,000 luminescence units) and increasing amounts of the cDNA for CB₁R-YFP; c and d, A_2A^A374-Rluc receptor (1 μg of cDNA; ∼100,000 luminescence units), D₂-GFP² receptor (3 μg of the cDNA; ∼6,000 fluorescence units), and increasing amounts of cDNA of CB₁-YFP receptor (8,000–18,000 fluorescence units). In each sample fluorescence or luminescence was measured before every experiment to confirm similar donor expressions while monitoring the increased acceptor expression. a, BRET¹ saturation curves for the A_2A-Rluc-D₂-YFP receptor pair (squares) and for the A_2A^A374-Rluc-D₂-YFP receptor pair (triangles) were obtained by monitoring the YFP fluorescence emission after coelenterazine H addition, with subtraction of the value obtained with cells expressing the same amount of donor. Data are expressed as means ± S.D. of five different experiments grouped as a function of the amount of BRET¹ acceptor. b, BRET¹ saturation curves for the A_2A-Rluc-CB₁-YFP receptor pair (triangles) and for the A_2A^A374-Rluc-CB₁-YFP receptor pair (squares) were obtained by monitoring the YFP fluorescence emission after coelenterazine H addition, with subtraction of the value obtained with cells expressing the same amount of donor. Data are expressed as means ± S.D. of five different experiments grouped as a function of the amount of BRET¹ acceptor. c, net SRET² was obtained by monitoring the YFP fluorescence emission after DeepBlueC addition, with subtraction of the value obtained with cells expressing the same amount of A_2A^A374-Rluc and D₂-GFP² receptors. SRET saturation curves (solid line) were obtained for the coupling of A_2A^A374-Rluc, D₂-GFP², and CB₁-YFP receptors and compared with the curve obtained for the coupling of A_2A-Rluc, D₂-GFP², and CB₁-YFP receptors (dotted line, see Fig. 1). SRET data are expressed as mean ± S.D. of five different experiments grouped as a function of the amount of SRET acceptor. d, BRET¹, BRET², and FRET were measured as indicated in Fig. 1 legend. Data are expressed as % of values obtained in cells expressing A_2A-Rluc, D₂-GFP², and CB₁-YFP (control, Fig. 1b) in mean ± S.E. of five independent experiments performed in duplicate. One-way ANOVA followed by Bonferroni test showed significant increases or decreases with respect to the control (*, p < 0.05; **, p < 0.01; ***, p < 0.005). Linear unmixing of the emission signals was applied to the data for BRET² and FRET values (e) and for YFP quantification in saturation curves (a and b). e, spectrum of a mixture of the following three peptides SAQEAQGNT, SAQEpSQGNT, and VLRRRRKRVN shows only one NCX between SAQEpSQGNT and VLRRRRKRVN at 2353.6 atomic mass units (see text). mBu, milli-BRET unit.

FIGURE 7.

A_2A-CB₁-D₂ receptor heteromerization in living cells treated with casein kinase 1/2 inhibitors. SRET² saturation experiments were performed 48 h post-transfection in cells expressing A_2A-Rluc receptor (1 μg of cDNA), D₂-GFP² receptor (3 μg of cDNA),and increasing amounts of CB₁-YFP receptor cDNA, treated with the casein kinase 1 inhibitor IC 261 (50 μm) and casein kinase 2 inhibitor TBAC (10 μm) as described under “Experimental Procedures.” In each sample fluorescence or luminescence was measured before every experiment to confirm similar donor expressions (∼100,000 luminescence units) and similar GFP² fluorescence (∼6,000 fluorescence units) while monitoring the increased acceptor expression (8,000–18,000 YFP fluorescence units). Net SRET² was obtained by monitoring the emission of YFP fluorescence after DeepBlueC addition, with subtraction of the value obtained with cells expressing the same amount of receptor Rluc and receptor GFP². SRET² saturation curves (solid lines) were compared with the curve obtained for the coupling of A_2A-Rluc, D₂-GFP², and CB₁-YFP receptors in cells not treated with casein kinase inhibitors (dotted line, see Fig. 1). SRET data are expressed as means ± S.D. of five different experiments grouped as a function of the amount of SRET acceptor.

FIGURE 8.

Molecular model of the A_2A-CB₁-D₂ receptor heteromer. a, schematic model of the heteromerization of A_2A (gold), CB₁ (red), and D₂ (cyan) receptors. Solid lines between TM5 and -6 symbolize IL3 of CB₁ (red line, 29 amino acids long) or D₂ (cyan line, 142 amino acids long) receptors, which were not modeled; solid lines after HX8 represent CT of CB₁ (red line) or A_2A (gold line), which were arbitrarily modeled as in squid rhodopsin; red spheres represent either phosphorylated Thr³²¹(IL3)–Ser³²²(IL3) or Thr⁴⁶⁷(CT)–Ser⁴⁶⁸(CT) of CB₁ or phosphorylated Ser³⁷⁴(CT) of A_2A; and blue half-circles represent either Arg²⁰⁵(5.66)–Arg²⁰⁶(5.67) of A_2A or the ²¹⁵(5.64)VLRRRRKRVN²²⁴ or ²⁶⁶NRRRVEAARR²⁷⁵(IL3) epitopes of D₂. b, lateral and cytoplasmic views of the computational model of the A_2A-CB₁-D₂ receptor heteromer. GFP fused to Cys⁴⁴³(CT) of the D₂ receptor (cyan surface) and YFP fused to Leu⁴⁷²(CT) of the CB₁ receptor (red surface) are shown. IL3 of CB₁ (red line) and D₂ (cyan line) receptors are shown in solid lines to illustrate their proximity. c, cytoplasmic view of the computational model of the A_2A-CB₁-D₂ receptor heteromer. CT of the CB₁ receptor is depicted in the following manner: amino acids Ser⁴¹⁴–Asn⁴³⁷ of (red tube ribbon) are modeled as in the crystal structure of squid rhodopsin, amino acids Asn⁴³⁷–Asp⁴⁶⁶ (not modeled) are shown as a red solid line to illustrate the position of Thr⁴⁶⁷–Ser⁴⁶⁸, and amino acids Ala⁴⁶⁹–Leu⁴⁷² (red solid line) are arbitrarily modeled to position YFP. CT of the A_2A receptor is depicted in the following manner: amino acids Ser³⁰⁵–Gly³²⁸ (golden tube ribbon) are modeled as in the crystal structure of squid rhodopsin; amino acids Ser³²⁹–Ser⁴¹² (not modeled) are shown as a yellow solid line, and phosphorylated Ser³⁷⁴ is shown as a red circle. Helices are shown as cylinders with the following color codes: TM4 in gray, TM5 in green, TM6 in blue, and the other helices in yellow for A_2A, in red for CB₁, and cyan for D₂ receptors.

FIGURE 9.

Agonist-induced ERK1/2 phosphorylation by the A_2A-D₂-CB₁ receptor heteromer. a and b, assays were performed 48 h post-transfection in cells expressing the indicated receptors (1.2 μg of cDNA of the A_2A or the A_2A^A205-A206 receptors, 1 μg of cDNA of the D₂, 0.8 μg of cDNA of the D_2S receptor, and 1 μg of cDNA of the CB₁, CB₁^A467-A468, or the CB^1A321-A322 receptors). Cells were treated for 5 min with 200 nm CGS 21680 (CGS), 1 μm quinpirole (Quinp), or both (CGS+Quinp) and ERK1/2 phosphorylation was determined as indicated under “Experimental Procedures.” The immunoreactive bands from four experiments performed in duplicate were quantified, and the values represent the mean ± S.E. of % of phosphorylation relative to the basal levels found in untreated cells. c, assays were performed in striatal slices from wild-type (WT) or CB₁ knock-out mice (CB₁-KO). The slices were treated for 10 min with 1 μm CGS 21680 (CGS), 1 μm quinpirole (quinpirole) or both, and ERK1/2 phosphorylation was determined as indicated under “Experimental Procedures.” The immunoreactive bands from four to eight slices obtained from five to nine animals were quantified, and values represent the mean ± S.E. of the % of phosphorylation relative to basal levels found in untreated slices. Significant differences respect to the wild-type mice were calculated by bifactorial ANOVA followed by post hoc Bonferroni's tests (**, p < 0.01; ***, p < 0.001).