μ and δ Opioid Receptors Are Differentially Desensitized by the Coexpression of β-Adrenergic Receptor Kinase 2 and β-Arrestin 2 in Xenopus Oocytes

Abstract

The Xenopus oocyte expression system was used to test the hypothesis that homologous opioid receptor desensitization results from receptor phosphorylation by G protein-coupled receptor kinases. Activation of ␦ (DOR), (MOR) opioid, or ␤ 2 -adrenergic receptors increased K ؉ conductance in oocytes coexpressing the G protein-gated inwardly rectifying K ؉ channel subunits GIRK1 and GIRK4, and the intrinsic rate of desensitization was small. Coexpression of ␤-adrenergic receptor kinase 2 (␤-ARK2) and ␤-arrestin 2 (␤-arr2) synergistically produced a rapid desensitization of both DOR and ␤2-adrenergic receptor signaling with a t1 ⁄2 < 4 min. ␤-ARK2 and ␤-arr2 more slowly desensitized MOR responses; a similar synergistic effect on MOR required 2-3 h of agonist treatment. DOR mutants lacking serine and threonine residues at the end of the cytoplasmic tail coupled effectively to GIRK channels but were insensitive to ␤-ARK2 and ␤-arr2. However, a DOR mutant having serine residues mutated to alanine in the third cytoplasmic loop was indistinguishable in coupling and desensitization from the wild type DOR. These studies establish that opioid receptors can be regulated by ␤-ARK2 and ␤-arr2 and that a portion of the COOH terminus of DOR enhances sensitivity to this modulation.Opioid agonists are the drugs of choice in the management of severe and chronic pain; however, continuous use of opioids can cause undesirable side effects including drug tolerance and addiction. The processes underlying tolerance are complex and involve learning mechanisms, compensatory changes in neuronal circuits, and desensitization of signal transduction mechanisms (1). Three classes of opioid receptors: , ␦, and , have been defined pharmacologically, shown to couple to G proteins of the G i /G o family, and cloned (2, 3). Previous studies have examined the molecular mechanisms underlying opiate tolerance. Chronic treatment of animals with opiates has not revealed a functionally significant change in opioid receptor number (4, 5) or mRNA levels (Ref. 6, but see also Refs. 7 and 8). However, opioid receptor desensitization has been consistently implicated as one of the molecular events accompanying tolerance (9). Continuous infusion of guinea pigs or rats with morphine results in an uncoupling of opioid receptors from associated G proteins as measured biochemically (10, 11). Recordings from single rat brain neurons of the locus coeruleus show a homologous desensitization to opioid-induced increases in potassium conductance after chronic morphine treatment (12). Prolonged morphine administration reduced the ability of DAMGO, 1 a selective opioid agonist, to stimulate [ 35 S]GTP␥S binding in distinct rat brain areas (13). Chronic morphine treatment of rats also led to a homologous (e.g. receptor-specific) desensitization of either ␦ or opioid receptor activity as measured by the ability of specific agonists to inhibit adenylyl cyclase in several brain structures (14).Termination of signaling of other G protein-coupled receptors results from agonist-depe...

Noncanonical GPCR signaling arising from a PTH receptor–arrestin–Gβγ complex

“…S3A). When PTHR was activated by M-PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), the diffusion of Gβγ remained unchanged (Fig. S3B).…”

“…The difference in duration of cAMP signaling is thought to depend on the capacity of M-PTH(1-28), like PTH , to form an unusually persistent high-affinity complex with PTHR that is independent of G-protein coupling, whereas M-PTH(1-14) forms a more conventional high-affinity complex that is transient and dependent on G-protein coupling (2,20,21). The interactions of HA-PTHR with Gβγ CFP and β-arrestins, weakly detectable under basal conditions, increased significantly within 5 min after challenge with M-PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) or M-PTH(1-28) (Fig. S1).…”

“…For these assays, Gβγ CFP was expressed in HEK293 cells stably expressing HA-tagged PTHR (HA-PTHR), and cell lysates were prepared at different time points after a short exposure to either M-PTH(1-28) or M-PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), modified analogs of PTH that induce longer or shorter cAMP generation and biological responses, respectively (20). The difference in duration of cAMP signaling is thought to depend on the capacity of M-PTH(1-28), like PTH , to form an unusually persistent high-affinity complex with PTHR that is independent of G-protein coupling, whereas M-PTH(1-14) forms a more conventional high-affinity complex that is transient and dependent on G-protein coupling (2,20,21).…”

“…In either case, the classical model predicts that arrestin negatively regulates the levels of the L-R*-G complex, providing the main mechanism by which the signal of a GPCR system is turned off (4,5). This model, primarily derived from the analysis of the behavior of the β 2 -adrenergic receptor (β 2 -AR) (4,5) and many other class 1 GPCRs (6)(7)(8)(9)(10)(11)(12), is taken to be universal for GPCR biology (13). Recent findings have challenged this view.…”

Agonist‐Induced Phosphorylation of the κ‐Opioid Receptor

“…Desensitization of the K-opioid receptor following acute agonist stimulation of transfected cells was blocked by the expression of a dominant negative G protein-coupled receptor kinase (Raynor et al, 1994), and desensitization of the 6-opioid receptor was enhanced by overexpression of the G protein-coupled receptor kinase 2 and 5 (Pei et al, 1995). Furthermore, in Xenopus oocytes, homologous agonist-induced opioid receptor desensitization required co-expression of a G protein-coupled receptor kinase and~-arrestin (Kovoor et al, 1997). In the brain, agonist-induced desensitization of the~-opioid receptor was shown to be reduced by a nonselective kinase inhibitor (Harris and Williams, 1991).…”