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Biochim Biophys Acta. 1997 Dec 12;1359(3):263-74.

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

Author information

1
Institute of Biology, National Centre for Scientific Research Demokritos, Athens, Greece. iro@cyclades.nrcps.ariadne-t.gr

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.

PMID:
9434132
DOI:
10.1016/s0167-4889(97)00097-9
[Indexed for MEDLINE]
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