Engineered G protein coupled receptors reveal independent regulation of internalization, desensitization and acute signaling

Abstract

Background: The physiological regulation of G protein-coupled receptors, through desensitization and internalization, modulates the length of the receptor signal and may influence the development of tolerance and dependence in response to chronic drug treatment. To explore the importance of receptor regulation, we engineered a series of Gi-coupled receptors that differ in signal length, degree of agonist-induced internalization, and ability to induce adenylyl cyclase superactivation. All of these receptors, based on the kappa opioid receptor, were modified to be receptors activated solely by synthetic ligands (RASSLs). This modification allows us to compare receptors that have the same ligands and effectors, but differ only in desensitization and internalization.

Results: Removal of phosphorylation sites in the C-terminus of the RASSL resulted in a mutant that was resistant to internalization and less prone to desensitization. Replacement of the C-terminus of the RASSL with the corresponding portion of the mu opioid receptor eliminated the induction of AC superactivation, without disrupting agonist-induced desensitization or internalization. Surprisingly, removal of phosphorylation sites from this chimera resulted in a receptor that is constitutively internalized, even in the absence of agonist. However, the receptor still signals and desensitizes in response to agonist, indicating normal G-protein coupling and partial membrane expression.

Conclusions: These studies reveal that internalization, desensitization and adenylyl cyclase superactivation, all processes that decrease chronic Gi-receptor signals, are independently regulated. Furthermore, specific mutations can radically alter superactivation or internalization without affecting the efficacy of acute Gi signaling. These mutant RASSLs will be useful for further elucidating the temporal dynamics of the signaling of G protein-coupled receptors in vitro and in vivo.

Figure 1

Design of RASSL variants. The design of each RASSL variant is shown on the left. Differences in amino acid sequences of the receptor variants are shown in the C-terminal alignments, right. Sequence derived from the MOR are dark, while KOR sequences are light. All mutated residues are underlined.

Figure 2

Agonist-induced signaling and internalization of Rog. (A) Maximum calcium response plotted as a function of spiradoline dose for cells transfected with Rog or the wild-type KOR and treated with dynorphin or spiradoline. (B) Confocal micrographs show representative internalization of GFP-tagged Rog receptors 1 h after treatment with 10 μM or 100 μM spiradoline. Dynorphin at 100 μM (far right) caused little internalization in this assay. (C) ELISA for FLAG-tagged cell-surface receptors shows dose-dependent loss of receptors from cell surface one h after spiradoline treatment. After a 1 μM dose of spiradoline, internalization is evident within 15 minutes. Data are expressed as a percentage of receptors detected on surface of untreated cells expressing Rog. Error bars represent SEM for three replicates.

Figure 3

Agonist-induced internalization is reduced in Rog-A, but not Rog-μ. (A) Confocal micrographs showing localization of GFP-tagged Rog (left), Rog-A (center), or Rog-μ (right) stably expressed in HEK293 cells and treated with 10 μM spiradoline for 1 h before fixation. (B) A dose-response ELISA shows less internalization of Rog-A in response to 1 h of spiradoline at doses of 0.3–100 μM spiradoline. After treatment with 1 μM spiradoline, Rog-A showed less internalization up to 1 h after treatment. (C) A dose-response ELISA shows more internalization of Rog-μ in response to 1 h of low doses of spiradoline ranging from 0.01 to 0.1 μM. After treatment with 1 μM spiradoline, there was no difference in the time course of internalization between Rog and Rog-μ. ELISA data are expressed as a percentage of receptors detected on surface of untreated cells expressing the same receptor.

Figure 4

Antagonist treatment increases cell-surface expression of Rog-μA. (A) ELISA comparing cell-surface expression of receptors stably expressed in HEK293 cell lines, either untreated (white bars) or treated for 18 h with the antagonist norBNI (10 μM, dark bars). Error bars represent SEM for three replicates. NorBNI significantly increased cell-surface expression of both Rog-μ and Rog-μA. OD, optical density. (B) Confocal micrographs showing that the localization of GFP-tagged Rog-μA is primarily intracellular in untreated cells (left), but the receptor moves to the membrane after 18 hours of antagonist treatment (right). There is relatively little change in Rog after norBNI treatment relative to untreated cells.

Figure 5

Desensitization of cAMP inhibition and superactivation of AC after pretreatment with spiradoline (A) Spiradoline (1 nM pretreatment) inhibited forskolin-induced cAMP formation in HEK293 cells transiently expressing Rog, Rog-A, Rog-μ, and Rog-μA. Data are expressed as inhibition of forskolin-induced cAMP. The baseline (0) represents maximum forskolin-induced cAMP accumulation in control cells. Pretreatment with 1 nM spiradoline for 10 min causes a shift in the dose-response curve for later spiradoline treatment in Rog and Rog-μA cells, but not Rog-A. Spiradoline pretreatment lowered the maximal response of Rog-μ to subsequent spiradoline treatment. (B) HEK293 cells transiently expressing receptors were treated 18 h with 10 nM spiradoline, and assayed for cAMP accumulation in response to a 15-min treatment with 10 μM forskolin. Spiradoline pretreatment significantly increased forskolin-induced cAMP in cells expressing KOR, Rog and Rog-A. Pretreatment of cells expressing Rog-μ and Rog-μA had not effect on response to forskolin. Data are expressed relative to the amount of cAMP accumulated after 10 μM forskolin treatment in cells pretreated with vehicle. Bars represent mean ± SEM for six replicates per condition. (C) HEK293 cells transiently expressing receptors were treated 18 h with 10 nM spiradoline, and assayed for loss of cell-surface expression by ELISA. Long-term spiradoline treatment significantly reduces cell-surface expression of all receptors, but the degree of internalization does not differ among different receptors. Bars represent mean ± SEM for three replicates per condition.