Opioid receptors from a lower vertebrate (Catostomus commersoni): sequence, pharmacology, coupling to a G-protein-gated inward-rectifying potassium channel (GIRK1), and evolution

Abstract

The molecular evolution of the opioid receptor family has been studied by isolating cDNAs that encode six distinct opioid receptor-like proteins from a lower vertebrate, the teleost fish Catostomus commersoni. One of these, which has been obtained in full-length form, encodes a 383-amino acid protein that exhibits greatest sequence similarity to mammalian mu-opioid receptors; the corresponding gene is expressed predominantly in brain and pituitary. Transfection of the teleost cDNA into HEK 293 cells resulted in the appearance of a receptor having high affinity for the mu-selective agonist [D-Ala2, MePhe4-Gly-ol5]enkephalin (DAMGO) (Kd = 0.63 +/- 0.15 nM) and for the nonselective antagonist naloxone (Kd = 3.1 +/- 1.3 nM). The receptor had negligible affinity for U50488 and [D-Pen2, D-Pen5]enkephalin (DPDPE), which are kappa- and delta-opioid receptor selective agonists, respectively. Stimulation of transfected cells with 1 microM DAMGO lowered forskolin-induced cAMP levels, an effect that could be reversed by naloxone. Experiments in Xenopus oocytes have demonstrated that the fish opioid receptor can, in an agonist-dependent fashion, activate a coexpressed mouse G-protein-gated inward-rectifying potassium channel (GIRK1). The identification of six distinct fish opioid receptor-like proteins suggests that additional mammalian opioid receptors remain to be identified at the molecular level. Furthermore, our data indicate that the mu-opioid receptor arose very early in evolution, perhaps before the appearance of vertebrates, and that the pharmacological and functional properties of this receptor have been conserved over a period of approximately 400 million years implying that it fulfills an important physiological role.

Figure 1

Primary structure of the C. commersoni μ-opioid receptor. The fish μ-opioid receptor sequence has been aligned with those of the rat μ- (11), κ- (9), and δ- (11) opioid receptors, and the rat ORL1 receptor (14), using the computer program pileup (Wisconsin Sequence Analysis Package, Version 8, Genetics Computer Group, Madison, WI). The seven putative membrane-spanning domains which are common to G-protein-coupled receptors are enclosed in boxes, and asterisks mark positions at which the C. commersoni and rat μ-receptor sequences are identical. In the fish sequence, consensus N-linked glycosylation sites are boxed, and potential targets for phosphorylation by either protein kinase A or C (see text) are high-lighted by white lettering on a black background.

Figure 2

Binding of opioid receptor ligands. Saturation binding experiments, with either the nonselective opioid receptor antagonist [³H]naloxone (A) or the μ-selective agonist [³H]DAMGO (B) to membranes prepared from HEK 293 cells expressing the C. commersoni μ-opioid receptor, were carried out as described. Specific binding was calculated by subtracting the nonspecific binding (determined in the presence of 10 μM naloxone) from the total binding determined using a range of concentrations of either [N-allyl-2,3-³H]naloxone (0.1–30 nM) or [³H]DAMGO (0.1–30 nM). (Insets) Scatchard plots calculated from the binding data shown.

Figure 3

Displacement of [³H]naloxone binding by DAMGO but not by either DPDPE or U50488. The binding of 10 nM [N-allyl-2,3-³H]naloxone to membranes prepared from HEK 293 cells expressing the C. commersoni μ-opioid receptor was determined in the presence of a range of concentrations of the μ-selective agonist DAMGO (0.1 nM to 10 μM; A), the δ-selective agonist DPDPE (0.1 nM to 10 μM; B), and the κ-selective agonist U50488 (0.1 nM to 3 μM; C).

Figure 4

Functional negative coupling to adenylate cyclase. Subtype-selective opioid receptor agonists were tested for their ability to inhibit forskolin-stimulated cAMP accumulation in HEK 293 cells expressing the C. commersoni μ-opioid receptor (filled bars) and in nontransfected cells (striped bars). Cells were incubated in the presence of 16 nM forskolin (FSK) alone or together with either DAMGO (1 μM), DAMGO (1 μM) plus naloxone (10 μM), naloxone (10 μM), U50488 (1 μM), or DPDPE (1 μM).

Figure 5

Functional coupling to mouse GIRK1. Dose-response curves are shown for the DAMGO-induced peak currents obtained from Xenopus oocytes expressing GIRK1 and either the C. commersoni μ-opioid receptor (•) or the rat μ-opioid receptor (□). Above these are displayed typical current traces recorded from an oocyte expressing only GIRK1 (Left) and from an oocyte expressing GIRK1 and the fish μ-opioid receptor (Right). Open bars indicate the period of time during which the perfusion medium was changed from ND-96 to hK; the solid bars denote the subsequent application of 1 μM DAMGO (bar A) and 300 μM BaCl₂ (bar B). Note that in oocytes expressing either GIRK1 alone or GIRK1 and a μ-opioid receptor, changing from a bathing medium with a low potassium concentration (2 mM; ND-96) to one with a high potassium concentration (96 mM; hK) induces an inward current due to endogenous potassium channels and basal activation of GIRK1. A subsequent response to DAMGO is only seen when a μ-opioid receptor is present. The application of BaCl₂, which specifically blocks GIRK1 currents, permits determination of the contribution of this channel to the total current response. (Bars: vertical, 60 nA; horizontal, 120 sec)

Figure 6

Tissue expression of the C. commersoni μ-opioid receptor gene. Shown are the results of a Northern blot analysis (A) and an RT-PCR experiment (B). The numbers on the left indicate the estimated transcript sizes (A) and the amplified cDNA fragment sizes (B). For details, see Materials and Methods.

Figure 7

A family of opioid receptor-like sequences exists in C. commersoni. The dendrogram shows the relationships of the partial sequences of five teleost fish opioid-like receptors to the rat μ- (11), κ- (9) and δ- (11) opioid receptors, and the rat ORL1 receptor (14); this was generated using the computer program pileup (Wisconsin Sequence Analysis Package).