Evidence is presented for the occurrence of a unique
opiate alkaloid-selective,
opioid peptide-insensitive binding site in N18TG2 mouse
neuroblastoma cells and in late passage hybrid F-11 cells, derived from N18TG2
neuroblastoma cells and rat dorsal root ganglion cells. Those cells lacked classical
opioid peptide-sensitive receptor subtypes, but contained [3H]
morphine and [3H]
diprenorphine binding sites with affinity for certain
opiate alkaloids but not for any endogenously occurring
opioid peptide or
peptide analog tested, including D-ala2-D-leu5-enkephalin (
DADLE), D-Ala2,N-Me-Phe4,Gly5-ol (
DAGO) and
dynorphin A(1-17). The binding site differed from hitherto described mu, delta and kappa neuronal
opioid receptors not only on the basis of
peptide insensitivity, but also on the basis of selectivity and affinities of
alkaloids. Saturation experiments with [3H]
morphine indicated the presence of a single site with Kd = 49 nM and Bmax = 1510 fmol/mg
protein. This novel binding site was not present in F-11 hybrid cells at early passage. Instead the hybrid cells contained conventional
opioid receptors (predominantly delta and also mu) capable of binding
DADLE and other
peptides as well as
opiate alkaloids. With additional passage (cell divisions) of the hybrid cells, during which a limited change occurred in mouse chromosome number, the
peptide-insensitive binding appeared and the
opioid peptide-binding (delta and
mu) receptors were lost reciprocally. Thus, expression of the
peptide-insensitive binding normally may be repressed when conventional
opioid receptors are expressed. The
peptide-insensitive
opiate binding site described here appears to correspond to the mu 3 receptor subtype, recently identified pharmacologically and functionally in several cell types of the immune system.(ABSTRACT TRUNCATED AT 250 WORDS)