Selective interactions of μ-opioid receptors with pertussis toxin-sensitive G proteins: involvement of the third intracellular loop and the c-terminal tail in coupling

Abstract

A cDNA encoding the rat mu-opioid receptor was expressed stably in a Rat-1 fibroblast cell line. Expression of this receptor was demonstrated with specific binding of the mu-opioid selective ligand [3H][D-Ala2,N-MePhe4,Gly5-ol]-enkephalin ([3H]DAMGO). In membranes of clone mu11 cells DAMGO produced a robust, concentration-dependent stimulation of basal high affinity GTPase activity. Cholera toxin-catalyzed [32P]ADP-ribosylation in membranes of this clone labelled a 40 kDa Gi family polypeptide(s) that was markedly enhanced by the addition of DAMGO. Antisera against Gi2alpha and Gi3alpha were both able to immunoprecipitate a [32P]-radiolabelled 40 kDa polypeptide(s) from DAMGO and cholera-toxin treated membranes of clone mu11, indicating that the mu-opioid receptor was able to interact effectively with both Gi2 and Gi3 in Rat-1 fibroblasts. A series of peptides derived from the delta-opioid receptor sequence were assessed for their ability to modify agonist-stimulated G protein activation and [3H] agonist binding to the receptor. In membranes from the clone mu11, specific binding of [3H]DAMGO was reduced by peptides corresponding to the NH2-terminal region of the third intracellular loop (i3.1) and the carboxyl-terminal tail (i4) of this receptor. Agonist stimulated GTPase activity and DAMGO dependent cholera toxin-catalyzed [32P]ADP-ribosylation were inhibited by peptides derived from the proximal (i3.1) and the distal portion (i3.3) of the third intracellular loop. Peptide i3.1 also inhibited DAMGO-stimulated [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTP-gammaS) binding in the same membranes. In contrast, peptides derived from the second intracellular loop were without any effect.

Rescuing the Traffic-Deficient Mutants of Rat μ-Opioid Receptors with Hydrophobic Ligands

“…During the course of the studies, two of these mutants, one with a deletion of five amino acids at the NH 2 -terminal of the i3 loop (⌬ 258 RLSKV 262 , i3-1) and another with a deletion at the proximal carboxyl tail (⌬ 344 KFCTR 348 , C-2), resulted in poor expression of these receptors in transfected HEK293 cells. Because previous studies have suggested that these two receptor domains are involved in the coupling to the G proteins (Georgoussi et al, 1997), the deletion of these motifs could generate constitutively active receptors and account for the poor expression levels. To determine the mechanism that was responsible for the low expression of the receptor mutants i3-1 and C-2, the contributions of the agonist-independent constitutive activation and the intracellular retention of mutant receptors were examined.…”

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

“…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.…”

Direct and Differential Interaction of β-Arrestins with the Intracellular Domains of Different Opioid Receptors

“…It has been known that the third intracellular loop (I3L) and the carboxyl terminus (CT) of GPCRs are crucial domains for receptor function, and this is also the case for opioid receptors. The third intracellular loop of opioid receptors has been suggested as a regulation target by calmodulindependent protein kinase II (Koch et al, 1997), in addition to its established role in G protein activation (Merkouris et al, 1996;Georgoussi et al, 1997). In contrast, the carboxyl terminus of opioid receptors seems to be more significantly involved in the modulation of receptor function by protein kinases and ␤-arrestins (Kovoor et al, 1997;Cheng et al, 1998;Appleyard et al, 1999), as well as in the receptor coupling with G proteins (Merkouris et al, 1996;Georgoussi et al, 1997).…”

“…The third intracellular loop of opioid receptors has been suggested as a regulation target by calmodulindependent protein kinase II (Koch et al, 1997), in addition to its established role in G protein activation (Merkouris et al, 1996;Georgoussi et al, 1997). In contrast, the carboxyl terminus of opioid receptors seems to be more significantly involved in the modulation of receptor function by protein kinases and ␤-arrestins (Kovoor et al, 1997;Cheng et al, 1998;Appleyard et al, 1999), as well as in the receptor coupling with G proteins (Merkouris et al, 1996;Georgoussi et al, 1997). The present work, with employment of glutathione S-transferase (GST) pull-down assay and surface plasmon resonance (SPR) technique, was thus designed to study in vitro the direct interaction of ␤-arrestins with those two functional domains, the third intracellular loop and the carboxyl terminus, of opioid receptors.…”