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. 2001 Jan 2;98(1):343-8.
doi: 10.1073/pnas.98.1.343.

Oligomerization of opioid receptors with beta 2-adrenergic receptors: a role in trafficking and mitogen-activated protein kinase activation

B A Jordan  1 , N Trapaidze, I Gomes, R Nivarthi, L A Devi
Affiliations

Oligomerization of opioid receptors with beta 2-adrenergic receptors: a role in trafficking and mitogen-activated protein kinase activation

B A Jordan et al. Proc Natl Acad Sci U S A. .

Abstract

G-protein-coupled receptors (GPCRs) have recently joined the list of cell surface receptors that dimerize. Dimerization has been shown to alter the ligand-binding, signaling, and trafficking properties of these receptors. Recent studies have shown that GPCRs heterodimerize with closely related members, resulting in the modulation of their function. In this study, we have attempted to determine whether members of GPCR superfamilies that couple to different families of G-proteins can associate and form oligomers. We chose the beta2 adrenergic receptor that couples to stimulatory G-proteins and delta & kappa opioid receptors that couple to inhibitory G-proteins. beta2 and delta receptors undergo robust agonist-mediated endocytosis, whereas kappa receptors do not. We find that when coexpressed, beta2 receptors can form heteromeric complexes with both delta and kappa receptors. This heterooligomerization does not significantly alter the ligand binding or coupling properties of the receptors. However, it affects the trafficking properties of the receptors. For example, we find that delta receptors, when coexpressed with beta2 receptors, undergo isoproterenol-mediated endocytosis. Conversely, beta2 receptors in these cells undergo etorphine-mediated endocytosis. However, beta2 receptors, when coexpressed with kappa receptors, undergo neither opioid- nor isoproterenol-mediated endocytosis. Moreover, these cells exhibit a substantial decrease in the isoproterenol-induced phosphorylation of mitogen-activated protein kinases. Taken together, these results provide direct evidence of heteromerization of GPCRs that couple to different types of G-proteins, which results in the modulation of receptor trafficking and signal transduction.

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Figures

Figure 1
Figure 1
(A) Myc-tagged δ or κ receptors interact with Flag-tagged β2 receptors to form heteromers. Immunoprecipitation of cell lysates from a mixture of HEK-293 cells individually expressing mycδ and Flagβ2 receptors (Flag β2 + mycδ) or Flagβ2 and mycκ receptors (Flagβ2 + mycκ) or cells coexpressing Flagβ2 and mycδ receptors (Flagβ2-mycδ) or Flagβ2 and mycκ receptors (Flagβ2-mycκ) was carried out with the use of anti-myc antibodies. Western blotting of the immunocomplexes with anti-Flag antibodies shows heteromers only in cells coexpressing the opioid and adrenergic receptors. IP, immunoprecipitation; WB, Western blotting. + DTT, immunoprecipitates treated with100 mM DTT in the sample buffer. (B) δ-β2 and κ-β2 heteromers exist on the cell surface. Cells individually expressing Flagβ2 or coexpressing Flagβ2-mycδ or Flagβ2-mycκ were incubated with anti-myc antibodies before solubilization and immunoprecipitation, as described under Materials and Methods. Western blotting of the immunocomplexes with anti-Flag antibodies shows the presence of the heteromers only in cells coexpressing both receptors. The 50-kDa band corresponds to a crossreactivity of the secondary Ab with the monoclonal Ab used for IP.
Figure 2
Figure 2
Agonist-mediated accumulation of cAMP in cells individually expressing epitope-tagged δ, κ, and β2 receptors or cells coexpressing δ-β2 or κ-β2 receptors. Cells were treated with indicated concentrations of etorphine (Left) or isoproterenol (Right) for 5 min at 37°C, and the intracellular cAMP level was measured by RIA as described under Materials and Methods. The amount of cAMP in cells without agonist treatment is taken as 100%. The data represent means ± SEM (n = 3–5).
Figure 3
Figure 3
Agonist-mediated internalization of receptors in HEK cells expressing δ, β2, δ-β2 (Left) or κ, β2, κ-β2 (Right) receptors. Cells were treated with indicated concentrations of etorphine for 60 min at 37°C, stained, and analyzed by ELISA, with the use of anti-Flag and anti-myc antibodies. The mean absorbance without agonist treatment is taken as 100%. The data represent means ± SEM (n = 5–7).
Figure 4
Figure 4
Agonist-mediated internalization of receptors in cells coexpressing κ-β2 or δ-β2 receptors. Cells were treated with indicated concentrations of 100 μM isoproterenol for 60 min at 37°C, stained, and analyzed by ELISA as described under Materials and Methods. The mean absorbance without agonist treatment is taken as 100%. Significant differences from untreated controls are indicated. **, P < 0.01, ***, P < 0.005 (n = 3–5).
Figure 5
Figure 5
Heterodimerization with κ receptors affects the level of β2 receptor-mediated phosphorylation of pMAP kinase. Cells expressing δ-β2 or κ-β2 receptors were treated with indicated concentrations of isoproterenol (A) or etorphine (B) for 5 min. The extent of phospho-MAP kinase (pMAPK) was determined by Western blotting analysis with antiphospho-MAP kinase antibody; standardization was with tubulin measured in the same blots, with the use of antitubulin antibody.
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