We have examined the endocytic trafficking of epitopetagged ␦ and opioid receptors expressed in human embryonic kidney (HEK) 293 cells. These receptors are activated by peptide agonists (enkephalins) as well as by the alkaloid agonist drugs etorphine and morphine. Enkephalins and etorphine cause opioid receptors to internalize rapidly (t1 ⁄2 ϳ 6 min) by a mechanism similar to that utilized by a number of other classes of receptor, as indicated by localization of internalized opioid receptors in transferrin-containing endosomes and inhibition of opioid receptor internalization by hypertonic media. Remarkably, morphine does not stimulate the rapid internalization of either ␦ or opioid receptors, even at high concentrations that strongly inhibit adenylyl cyclase. These data indicate that agonist ligands, which have similar effects on receptor-mediated signaling, can have dramatically different effects on the intracellular trafficking of a G protein-coupled receptor.Opioid receptors constitute a class of G protein-coupled receptors that mediate the effects of endogenously produced opioid peptides in the central and peripheral nervous systems. An interesting feature of these receptors is that they are activated both by native peptides and by structurally distinct non-peptide alkaloid ligands (1). Following activation, opioid receptors are regulated by multiple mechanisms, which modulate the functional plasticity of the endogenous opioid system and contribute to the development of opiate tolerance and dependence (2-5).Previous studies have described two distinguishable processes of opioid receptor regulation, termed desensitization and down-regulation (3, 4, 6 -9). Both desensitization and downregulation of opioid receptors are stimulated by peptide and alkaloid agonists (4, 6). However, individual agonists may have substantially different effects on the rapid regulation of opioid receptors (10 -12). We have observed that opioid receptors are regulated by a process of rapid internalization that exhibits a remarkable degree of agonist specificity not observed previously in studies of the intracellular trafficking of other receptors. EXPERIMENTAL PROCEDURES Construction and Expression of Epitope-tagged ␦ and OpioidReceptors-cDNAs encoding murine ␦ (13) and (14) opioid receptors were epitope-tagged in the amino-terminal extracellular domain utilizing a shuttle vector containing a signal-FLAG cassette (kindly provided by Drs. Jeff Reagan and Brian Kobilka) (15) and subcloned into pcDNA3 (Invitrogen) for transfection. The structure of each mutant cDNA was confirmed by dideoxy sequencing (Sequenase, U. S. Biochemical Corp.).Human embryonic kidney (HEK) 1 293 cells (ATCC) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (University of California San Francisco Cell Culture Facility) and transfected using calcium phosphate coprecipitation (16). Stably transfected cells were isolated following neomycin selection (Geneticin, Life Technologies, Inc.) as described previously (17), and satur...

Patients' preoperative expectations regarding rotator cuff repair are associated with their actual self-assessed outcome. Variations in patient expectations may help to explain divergent results in published series as well as among various patient populations.

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The mouse agouti coat color gene encodes a novel paracrine signaling molecule whose pulsatile expression produces a characteristic pattern of banded pigment in individual hairs. Several spontaneous agouti alleles produce adult-onset obesity and diabetes, and have provided important single-gene animal models for alterations in energy metabolism. Utilizing linkage groups conserved between mice and humans, we have cloned the human homolog of the mouse agouti gene from a human chromosome 20 yeast artificial chromosome known to contain S-adenosyl homocysteine hydrolase (AHCY). The human agouti gene, named Agouti Signaling Protein (ASP), encodes a 132 amino acid protein, the mRNA for which is expressed in testis, ovary, and heart, and at lower levels in liver, kidney, and foreskin. As predicted by the interactions of mouse agouti with the extension gene (which encodes the melanocyte receptor for alpha-melanocyte stimulating hormone [alpha-MSH]), expression of ASP in transgenic mice produces a yellow coat, and expression of ASP in cell culture blocks the alpha-MSH-stimulated accumulation of cAMP in mouse melanoma cells. The localization of ASP relative to other loci on chromosome 20 excludes it as a candidate for the MODY1 locus, a gene responsible for one form of early-onset non-insulin-dependent diabetes mellitus or maturity-onset diabetes of the young. The expression of ASP in human tissues suggests a function for agouti homologs in species that do not exhibit the characteristic phenotype of banded hairs.

Prognostic Level I. See Instructions to Authors for a complete description of levels of evidence.

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