Ligand-Selective Activation of μ-Opioid Receptor: Demonstrated with Deletion and Single Amino Acid Mutations of Third Intracellular Loop Domain

Abstract

The mechanism for the differential regulation of the -opioid receptor by agonists is investigated by identifying the receptor domains used to define the relative efficacies of three -opioid with Ala reduced the potency of DAMGO but not that of morphine PL017. Meanwhile, mutation of Thr 279 to Ala increased the potencies of morphine and PL017 but not that of DAMGO. The I278A mutation decreased the DAMGO coupling efficiency but increased the PL017 coupling efficiency. The R280A mutation resulted in the increase in PL017 potency and coupling efficiency without altering those of DAMGO and morphine. Thus, these mutation studies suggested that the activation of -opioid receptor and interaction between the critical domains such as RRITR within third intracellular loop and the G proteins are agonist-selective.Opioid receptors are members of the G protein-coupled receptors (GPCRs) (Wess, 1998). Mutational analyses of many members of GPCR family provide evidence that the second and third intracellular loops and the C-terminal tails are involved in the receptor/G protein coupling (for review, see Helmreich and Hofmann, 1996). However, the consensus sequence in the receptor/G protein coupling has yet to be identified. Possibly, in addition to its primary sequence, a secondary structure of the receptor, i.e., the amphipatic ␣-helix is a determinant for G proteins and receptor interaction. Sequence alignment revealed that BBXXB (B stands for basic amino acids and X is any amino acid) sequence was commonly found in the regions of GPCRs critical for G protein interaction. Detailed studies further suggested that the basic amino acids in BBXXB motif are crucial for activating Gprotein. In the human platelet-activating factor (PAF) receptor, mutation in the BBXXB motif resulted in mutants with low-affinity binding for PAF and less effective in mediating phosphatidylinositol hydrolysis (Parent et al., 1996). Use of peptides corresponding to the third intracellular loop of the ␦-opioid receptor, Georgoussi et al. (1997) and Merkouris et al. (1996) demonstrated the importance of this motif in the opioid receptor/G protein interaction. The basic amino acids within this motif have been suggested to form a polar pocket interacting with motifs in other domains of the GPCR, such as the conserved (D/E)RY motif at the N terminus of the second intracellular loop, thus stabilizing the receptor in the inactive states. Binding of the agonist results in transmembrane (TM) movement, thus disrupting such interaction and resulted in the receptor activation. This hypothesis is supported by studies with the mutation of amino acids within this region and the Ala insertion studies that resulted in constitutive activities of the muscarinic receptor (Liu et al

Agonist-selective signaling is determined by the receptor location within the membrane domains

“…The deletion of the 276 RRITR 280 sequence from MOR (I35) resulted in decreased interaction with G␣ and the inability to mediate AC inhibition (28). The weaker interaction between I35 and G␣i2 was confirmed by the lesser amount of MOR or G␣i2 coimmunoprecipitated by each other (Fig.…”

“…N2A cells with HA-MOR and MEF cells were cultured in DMEM with 10% FBS with or without 200 ng/ml G418. MOR mutants, with 276 RRITR 280 sequence in the third intracellular loop (I35) or the carboxyl tail sequence after Ser-363 deleted (363D), were generated, as described (28,30). Sense and antisense G␣i2 in pCDNA3 were transfected to HEK293 cells by using Effectene (Qiagen).…”

Multiple Signaling States of G-Protein-Coupled Receptors

“…Deletion of the four to five amino acid clusters resulted in differential effects on the affinities of the agonists and antagonists and also on the potencies and coupling efficiencies of the three opioid agonists. Thus, these mutational studies suggested that the activation of -opioid receptor and interaction between the critical domains within the third intracellular loop and the G-proteins are agonist-selective (Chaipatikul et al, 2003). The study also suggested that differences in the agonist response were due to the relative spatial orientation of the amino acids within the intracellular domain after agonist binding in determining the efficiency of the receptor to activate the G-proteins.…”

Expression of the Third Intracellular Loop of the δ-Opioid Receptor Inhibits Signaling by Opioid Receptors and Other G Protein-Coupled Receptors

“…Our attempts were specifically focused in the third loop of ␦-OR, because this region was implicated in direct G protein binding and activation (Chan et al, 1995;Georgoussi et al, 1997), arrestin binding (DeGraff et al, 2002), and ligand-selective activation (Chaipatikul et al, 2003). We found that cellular expression of a 23-amino acid polypeptide derived from the i3L of ␦-OR was able to target the opioid receptor as assessed by coprecipitation studies.…”

“…1A). The i3L domain has been shown to be critical for G protein coupling and activation (Merkouris et al, 1996; and to participate in receptor desensitization (Cen et al, 2001) and other aspects of signaling (Megaritis et al, 2000;Chaipatikul et al, 2003).…”