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.
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.
Georgoussi
Z
Z
Institute of Biology, National Centre for Scientific Research Demokritos, Athens, Greece. iro@cyclades.nrcps.ariadne-t.gr
Merkouris
M
M
Mullaney
I
I
Megaritis
G
G
Carr
C
C
Zioudrou
C
C
Milligan
G
G
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002772:Cholera Toxin; D018076:DNA, Complementary; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004745:Enkephalins / Q000378:metabolism; D005347:Fibroblasts; D020558:GTP Phosphohydrolases / Q000378:metabolism; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000494:pharmacology; D037342:Pertussis Toxin; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism*; D051381:Rats; D017450:Receptors, Opioid, mu / Q000819:agonists / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D018076:DNA, Complementary; D004745:Enkephalins; D009842:Oligopeptides; D011518:Proto-Oncogene Proteins; D017450:Receptors, Opioid, mu; D010566:Virulence Factors, Bordetella; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D006153:Guanosine Diphosphate; D006165:Guanylyl Imidodiphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D002772:Cholera Toxin; D011065:Poly(ADP-ribose) Polymerases; D037342:Pertussis Toxin; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; C498017:Gnai2 protein, rat