Cell surface targeting of mu-delta opioid receptor heterodimers by RTP4

Abstract

Mu opioid receptors are G protein-coupled receptors that mediate the pain-relieving effects of clinically used analgesics, such as morphine. Accumulating evidence shows that mu-delta opioid heterodimers have a pharmacologic profile distinct from those of the mu or delta homodimers. Because the heterodimers exhibit distinct signaling properties, the protein and mechanism regulating their levels have significant effects on morphine-mediated physiology. We report the characterization of RTP4, a Golgi chaperone, as a regulator of the levels of heterodimers at the cell surface. We show that the association with RTP4 protects mu-delta receptors from ubiquitination and degradation. This leads to increases in surface heterodimer levels, thereby affecting signaling. Thus, the oligomeric organization of opioid receptors is controlled by RTP4, and this governs their membrane targeting and functional activity. This work is the first report of the identification of a chaperone involved in the regulation of the biogenesis of a family A GPCR heterodimer. The identification of such factors as RTP4 controlling dimerization will provide insight into the regulation of heterodimers in vivo. This has implications in the modulation of pharmacology of their endogenous ligands, and in the development of drugs with specific therapeutic effects.

Fig. 1.

Coexpression of μ and δ receptors leads to loss of cell surface μ receptors in HEK293 cells. (A) Cell surface expression of receptors shows that coexpression with myc-tagged δ receptors induces a large retention of FLAG-tagged μ receptors in an intracellular compartment. Coexpression of HA-tagged α_2AR with μ receptor or δ receptor with FLAG-tagged κ does not lead to intracellular retention supporting the specificity of μ-δ interactions. (B) Quantitation of cell surface expression of receptors shows that coexpression with myc-tagged δ receptors but not with HA-tagged α_2A receptors induces a large decrease in FLAG-tagged μ receptor expression. Coexpression of δ receptor does not alter the cell surface expression of FLAG-tagged κ receptors in HEK293 cells. (C) Quantitation of cell surface expression of δ receptors cotransfected with μ receptors in Neuro2A cells. Values are expressed as mean ± standard error of the mean (SEM) (n = 3).

Fig. 2.

Coexpression of μ and δ receptors leads to intracellular rentention of both receptors in the Golgi apparatus. Immunofluoresence images of Neuro2A cells cotransfected with μ (Left) and δ receptors (Middle). Labeling with anti-syntaxin 6 antibody (Middle) as well as TGN38 (Lower) shows that intracellular μ-δ receptors colocalize with the marker for Golgi apparatus and not with that of ER compartment (anti-calnexin antibody; Upper).

Fig. 3.

Expression of RTP4 prevents the retention of μ-δ receptors in HEK293 cells. (A) Quantitation of cell surface expression of μ receptors cotransfected with δ receptors without or with RTP4. Expression of increasing amounts of RTP4 cDNA (1 or 2 μg) leads to a concomitant increase in the level of cell surface μ expression. Data are given as mean ± SEM (n = 3). (B) Immunofluoresence labeling of μ (green) and δ (red) receptors transfected without (Upper) and with (Lower) RTP4. The intracellular accumulation of both receptors (yellow, Upper Right) is decreased when RTP4 is coexpressed, leading to increased cell surface localization of both μ and δ receptors (Lower).

Fig. 4.

RTP4 interacts with μ and δ receptors. (A) Western blotting analysis shows that RTP4 coimmunoprecipitates with full-length (μ FL) or Δ24 but not with Δ29- and Δ45-truncated μ receptors. In panels A, B and D cells were incubated with antibodies to the epitope tag before the lysis step so as to enable the isolation of cell surface receptors. (B) Quantitation of the coimmunoprecipitation of RTP4 with mutant μ receptors. The data with wild type μ receptors is taken as 1.0; data represents mean ± SEM (n = 3). P3 represents cells transfected with control plasmid, pCDNA3. (C) Western blot analysis shows that increased myc-tagged δ receptors are detected after HA-tagged μ immunoprecipitation in the presence of RTP4. (D) Quantitation of coimmunoprecipitation data, expressed as mean ± SEM (n = 3). (E) Western blot analysis shows the level of ubiquitination of μ receptors when coexpressed with δ receptors; coexpression of RTP4 leads to a decrease in the extent of ubiquitination. (F) Quantification of the ubiquitination data, expressed as mean ± SEM (n = 3). All signals were normalized to the value with μ alone taken as 1.0.

Fig. 5.

Functional impact of RTP4 expression. (A) Coexpression with RTP4 leads to decreased degradation of μ-δ receptors as determined by quantification of HA-tagged μ receptors coexpressed with δ receptors without or with RTP4 treated without or with 50 μM MG132 at 37°C for 3 h before harvesting. (B) Coexpression with RTP4 leads to alteration in μ-δ signaling, as detected by the pCRE-SEAP assay. Coexpression of RTP4 leads to a decrease in signaling by DAMGO, as demonstrated by the decreased efficacy of DAMGO. Data are expressed as mean ± SEM (n = 3). (C) Whole cell binding in SKNSH cells transfected with control or RTP4-directed siRNA. The receptor levels were measured in cells 48 h after siRNA transfection using ³H-DAMGO (for μ) or ³H-Deltorphin (for δ). Data are given as mean ± SEM (n = 3), **, P < 0.001 and *, P < 0.05 versus control siRNA. (D) A model for the role of RTP4 in modulating opioid receptor levels. (Left) In the absence of RTP4, μ-δ heterodimers are largely retained in the Golgi apparatus (GA) and eventually routed to degradation pathways. (Right) In the presence of RTP4, the heterodimers are stabilized and routed to the membrane, leading to a reduced population of receptor homodimers.