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

Biochim Biophys Acta. 1997 Dec 12;1359(3):263-74. doi: 10.1016/s0167-4889(97)00097-9.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cell Line
  • Cell Membrane / metabolism
  • Cholera Toxin
  • DNA, Complementary
  • Enkephalin, Ala(2)-MePhe(4)-Gly(5)-
  • Enkephalins / metabolism
  • Fibroblasts
  • GTP Phosphohydrolases / metabolism
  • GTP-Binding Protein alpha Subunit, Gi2
  • GTP-Binding Protein alpha Subunits, Gi-Go*
  • GTP-Binding Proteins / chemistry*
  • GTP-Binding Proteins / metabolism*
  • Guanosine 5'-O-(3-Thiotriphosphate) / metabolism
  • Guanosine Diphosphate / pharmacology
  • Guanylyl Imidodiphosphate / pharmacology
  • Molecular Sequence Data
  • Oligopeptides / chemical synthesis
  • Oligopeptides / pharmacology
  • Pertussis Toxin*
  • Poly(ADP-ribose) Polymerases / metabolism
  • Proto-Oncogene Proteins / chemistry*
  • Proto-Oncogene Proteins / metabolism*
  • Rats
  • Receptors, Opioid, mu / agonists
  • Receptors, Opioid, mu / metabolism*
  • Virulence Factors, Bordetella / pharmacology*


  • DNA, Complementary
  • Enkephalins
  • Oligopeptides
  • Proto-Oncogene Proteins
  • Receptors, Opioid, mu
  • Virulence Factors, Bordetella
  • Enkephalin, Ala(2)-MePhe(4)-Gly(5)-
  • Guanosine Diphosphate
  • Guanylyl Imidodiphosphate
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • Cholera Toxin
  • Poly(ADP-ribose) Polymerases
  • Pertussis Toxin
  • GTP Phosphohydrolases
  • GTP-Binding Proteins
  • GTP-Binding Protein alpha Subunit, Gi2
  • GTP-Binding Protein alpha Subunits, Gi-Go
  • Gnai2 protein, rat