The binding and internalization of radioiodinated and fluorescent and ␦ opioid peptides in mammalian cells were quantitatively studied by biochemical techniques and directly visualized by confocal microscopy. The labeled peptides were prepared by inserting either a 125 IBolton-Hunter group or a fluorescent probe into the C-terminal part of 5-aminopentylamide derivatives of deltorphin-I and [Lys 7 ]dermorphin. The purified derivatives kept most of their specificity and selectivity toward ␦ and opioid receptors, respectively. Biochemical and confocal microscopy data showed that both and ␦ opioid peptides were internalized in mammalian cells transfected with the corresponding opioid receptor according to a receptor-mediated mechanism. The internalization process was time-and temperature-dependent and was completely blocked by the endocytosis inhibitor phenylarsine oxyde. Internalization of both ␦ and ligands occurred from a single large cap at one pole of the cell, indicating that polymerization of ligandreceptor complexes preceeded internalization. Finally, green and red fluorescent analogues of deltorphin-I and [Lys 7 ]dermorphin, respectively, were found to internalize through partly distinct endocytic pathways in cells co-transfected with and ␦ receptors, suggesting that each of these receptors interacts with distinct proteins mediating intracellular sorting and trafficking.The pharmacological, behavioral, and binding properties of , ␦, and opioid receptors have been extensively studied. By comparison, much less is known about the intracellular routing and addressing of opioid receptors either before or after agonist binding. Yet, understanding the cellular regulation of this class of receptors is of prime importance, since it is at least partly involved in the mechanisms of tolerance and physical dependence (1). There is a considerable amount of in vitro pharmacological evidence to suggest that both (2, 3) and ␦ (4 -7) opioid receptors may undergo rapid down-regulation following exposure to agonists. Whether, and under which condition, such down-regulation involves internalization of receptor-ligand complexes remains a matter of debate. Thus, while biochemical studies have reported on either the occurrence (5, 8) or absence (9) of internalization of the tritiated enkephalin agonist [3 H]DAla, D-Leu-enkephalin in cultured neuroblastoma cells, morphological studies clearly failed to observe internalization of a fluorescent derivative of enkephalin in the same cell system (10 -12). More recently, confocal microscopic studies carried out on transfected cells have shown a rapid endocytosis of (13,14) and ␦ (13) antigenic epitope-tagged receptors following exposure to enkephalins, but not to morphine. Whether this endocytosis occurs in conjunction with that of the bound ligand, however, remains unclear. In the present study, we have reinvestigated the fate of receptor-bound opioid peptides using newly developed radioactive and fluorescent derivatives of the selective and ␦ opioid agonists dermorphin and deltorphin.D...

The expression of the 3 currently known neurotensin receptors was studied in human cancer cells of prostatic, colonic or pancreatic origin by means of RT-PCR analysis and binding experiments. All the cells selected for this work have been shown to exhibit a growth response to neurotensin. We found that the 7 transmembrane domain, levocabastine insensitive receptor (NTR1) is expressed in most but not all of the cells studied whereas the 7 transmembrane domain, levocabastine sensitive receptor (NTR2) is present in none of these cells. The 100 kDa-type I neurotensin receptor (NTR3) is expressed in all the cells assayed. Moreover, we demonstrated that neurotensin can stimulate the growth of CHO cells stably transfected with the NTR3. Taken together, our results strongly suggest that the NTR3 subtype could be involved in the growth response of human cancer cells to neurotensin.

The inhibitory effect of the neuropeptide somatostatin on the expression of growth hormone was measured by quantitative polymerase chain reaction in the pituitary cell line AtT-20. We demonstrate that this effect is dependent on the internalization of somatostatin-receptor complexes and that it is totally independent from the peptide-induced inhibition of adenylate cyclase. Indeed, the inhibitory effect of the peptide on growth hormone mRNA levels was totally insensitive to pertussis toxin treatment but was totally abolished under conditions which block somatostatin receptor internalization. Comparative confocal microscopic imaging of fluorescent somatostatin sequestration and fluorescence immunolabeling of sst1, sst2A, and sst5 receptors suggests that sst2A is most probably responsible of the inhibitory effect of somatostatin on growth hormone expression.