Coupling efficacy and selectivity of the human mu-opioid receptor expressed as receptor-Galpha fusion proteins in Escherichia coli.

Two constructs encoding the human micro-opioid receptor (hMOR) fused at its C terminus to either one of two Galpha subunits, Galpha(o1) (hMOR-Galpha(o1)) and Galpha(i2) (hMOR-Galpha(i2)), were expressed in Escherichia coli at levels suitable for pharmacological studies (0.4-0.5 pmol/mg). Receptors fused to Galpha(o1) or to Galpha(i2) maintained high-affinity binding of the antagonist diprenorphine. Affinities of the micro-selective agonists morphine, [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO), and endomorphins as well as their potencies and intrinsic activities in stimulating guanosine 5'-O-(3-[(35)S]thiotriphosphate) ([(35)S]GTPgammaS) binding were assessed in the presence of added purified Gbetagamma subunits. Both fusion proteins displayed high-affinity agonist binding and agonist-stimulated [(35)S]GTPgammaS binding. In the presence of Gbetagamma dimers, the affinities of DAMGO and endomorphin-1 and -2 were higher at hMOR-Galpha(i2) than at hMOR-Galpha(o1), whereas morphine displayed similar affinities at the two chimeras. Potencies of the four agonists in stimulating [(35)S]GTPgammaS binding at hMOR-Galpha(o1) were similar, whereas at hMOR-Galpha(i2), endomorphin-1 and morphine were more potent than DAMGO and endomorphin-2. The intrinsic activities of the four agonists at the two fusion constructs were similar. The results confirm hMOR coupling to Galpha(o1) and Galpha(i2) and support the hypothesis of the existence of multiple receptor conformational states, depending on the nature of the G protein to which it is coupled.

Internalization and recycling of delta-opioid receptor are dependent on a phosphorylation-dephosphorylation mechanism.

Internalization, recycling, and resensitization of the human delta-opioid receptor (hDOR) were studied in the neuroblastoma cell line SK-N-BE, endogenously expressing this receptor. Conventional and confocal fluorescence microscopy observations, corroborated by Scatchard analysis, indicated that after a 100 nM Eto treatment, 60 to 70% of hDOR were rapidly internalized (t(1/2) < 15 min). This agonist-triggered internalization was reversible for a treatment not exceeding 1 h and became irreversible for prolonged treatment (4 h), leading probably to the degradation and/or down-regulation of the receptor. The rapid internalization of hDOR was totally blocked in the presence of heparin, known as an inhibitor of G protein-coupled receptor kinases (Benovic et al., 1989), a result indicating that phosphorylation by these kinases is a critical step in desensitization (Hasbi et al, 1998) and internalization of hDOR (present study) in SK-N-BE cell line. Blockade of internalization by agents not interferring with phosphorylation, as hypertonic sucrose or concanavalin A, also blocked the resensitization (receptor functional recovering) process. Furthermore, blockade of dephosphorylation of the internalized hDOR by okadaic acid totally suppressed its recycling to the plasma membrane and its subsequent resensitization. These results indicate that regulatory events leading to desensitization, internalization, and recycling in a functional state of hDOR involve phosphorylation by a G protein-coupled receptor kinase, internalization via clathrin-coated vesicles, and dephosphorylation by acid phosphatases.

Expression of delta, kappa and mu human opioid receptors in Escherichia coli and reconstitution of the high-affinity state for agonist with heterotrimeric G proteins.

Human opioid receptors of the delta, mu and kappa subtypes were successfully expressed in Escherichia coli as fusions to the C-terminus of the periplasmic maltose-binding protein, MBP. Expression levels of correctly folded receptor molecules were comparable for the three subtypes and reached an average of 30 receptors.cell-1 or 0.5 pmol.mg-1 membrane protein. Binding of [3H]diprenorphine to intact cells or membrane preparations was saturatable, with a dissociation constant, KD, of 2.5 nM, 0.66 nM and 0.75 nM for human delta, mu and kappa opioid receptors (hDOR, hMOR and hKOR, respectively). Recombinant receptors of the three subtypes retained selectivity and nanomolar affinity for their specific antagonists. Agonist affinities were decreased by one to three orders of magnitude as compared to values measured for receptors expressed in mammalian cells. The effect of sodium on agonist binding to E. coli-expressed receptors was investigated. Receptor high-affinity state for agonists was reconstituted in the presence of heterotrimeric G proteins. We also report affinity values of endomorphins 1 and 2 for mu opioid receptors expressed both in E. coli and in COS cells. Our results confirm that opioid receptors can be expressed in a functional form in bacteria and point out the advantages of E. coli as an expression system for pharmacological studies.

Heterologous expression of G-protein-coupled receptors: human opioid receptors under scrutiny.

G-protein-coupled receptors whose topology shows seven transmembrane domains form the largest known family of receptors involved in higher organism signal transduction. Despite increasing knowledge on the functioning mechanisms of these receptors, almost no structural data are available but only a few models. Structural studies using a wide range of physical and biochemical techniques may require fairly large (up to several milligrams) amounts of purified protein. Since such quantities are not naturally available, overexpression is prerequisite. Heterologous expression systems are then assayed for maximal production of a protein facsimile. Heterologous systems may also provide interesting alternatives for receptor functional studies in a different cellular context. Opioid receptors will be used as an example to discuss aspects related to the choice and suitability of several different expression systems for the intended analysis of G-protein-coupled receptor properties. General implications will be outlined.

Desensitization of the delta-opioid receptor correlates with its phosphorylation in SK-N-BE cells: involvement of a G protein-coupled receptor kinase.

Phosphorylation of G protein-coupled receptors is considered an important step during their desensitization. In SK-N-BE cells, recently presented as a pertinent model for the studies of the human delta-opioid receptor, pretreatment with the opioid agonist etorphine increased time-dependently the rate of phosphorylation of a 51-kDa membrane protein. Immunological characterization of this protein with an antibody, raised against the amino-terminal region of the cloned human delta-opioid receptor, revealed that it corresponded to the delta-opioid receptor. During prolonged treatment with etorphine, phosphorylation increased as early as 15 min to reach a maximum within 1 h. Phosphorylation and desensitization of adenylyl cyclase inhibition paralleled closely and okadaic acid inhibited the resensitization, a result strongly suggesting that phosphorylation of the delta-opioid receptor plays a prominent role in its rapid desensitization. The increase in phosphorylation of the delta-opioid receptor, as well as its desensitization, was not affected by H7, an inhibitor of protein kinase A and protein kinase C, but was drastically reduced by heparin or Zn2+, known to act as G protein-coupled receptor kinase (GRK) inhibitors. These results are the first to show, on endogenously expressed human delta-opioid receptor, that a close link exists between receptor phosphorylation and agonist-promoted desensitization and that desensitization involves a GRK.