Title: What's All the RAVE about Receptor Internalization?
Abstract: For some time, it has been assumed that the general role receptor endocytosis plays in G protein–coupled receptor (GPCR) actions is in the regulation of signal transduction and cell surface receptor expression. Recent studies have suggested that, in some cases, GPCR-mediated endocytosis may also be important for mediating other biological actions of receptor occupancy. These other actions influenced by receptor endocytosis may include physiologic responses such as tolerance, dependence, and even the therapeutic actions of various drugs (see 7Roth B.L. Willins D.L. Kroeze W.K Drug Alcohol Depend. 1998; 51: 73-85Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, for a recent review). For GPCRs in general, activation is usually followed by a cascade of events that includes receptor phosphorylation, desensitization, and internalization. It has been clear for some time, however, that efficacy does not always directly correlate with receptor phosphorylation, desensitization, and internalization (5Keith D.E. Murray S.R. Zaki P.A. Chu P.C. Lissin D.V. Kang L. Evans C.J. von Zastrow M J. Biol. Chem. 1996; 271: 19021-19024Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, 10Yu Y. Zhang L. Yin X. Sun H. Uhl G.R. Wang J.B J. Biol. Chem. 1997; 272: 28869-28874Crossref PubMed Scopus (197) Google Scholar; see figure). In some cases, even antagonists, which cannot activate receptors, cause GPCR internalization (9Willins D.L. Berry S.A. Alsayegh L. Backstrom J.R. Sanders-Bush E. Roth B.L Neuroscience. 1999; 91: 599-606Crossref PubMed Scopus (126) Google Scholar). With these findings as a backdrop8Whistler J.L. Chuang H.-H. Chu P. Jan L.Y. von Zastrow M Neuron. 1999; 23 (this issue,): 737-746Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar present intriguing data in this issue of Neuron which suggest that the differential biological actions of drugs may depend, in part, on their abilities to induce endocytosis. They use μ opioid receptors, which mediate many of the analgesic, rewarding, and addictive properties of opioids (6Matthes H.W. Maldonado R. Simonin F. Valverde O. Slowe S. Kitchen I. Befort K. Dierich A. Le Meur M. Dolle P. et al.Nature. 1996; 383: 819-823Crossref PubMed Scopus (1407) Google Scholar), as a model system in which to probe the consequences of GPCR endocytosis. In their first experiments8Whistler J.L. Chuang H.-H. Chu P. Jan L.Y. von Zastrow M Neuron. 1999; 23 (this issue,): 737-746Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar confirm previous observations (5Keith D.E. Murray S.R. Zaki P.A. Chu P.C. Lissin D.V. Kang L. Evans C.J. von Zastrow M J. Biol. Chem. 1996; 271: 19021-19024Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar) that even though morphine, a highly addictive opioid, can activate μ receptors, it does not readily induce internalization. Other drugs that also activate μ receptors, such as DAMGO (an enkephalin analog) and methadone, cause internalization of μ receptors (see figure). To determine if the differential effects of these drugs on internalization were due to differential receptor activation8Whistler J.L. Chuang H.-H. Chu P. Jan L.Y. von Zastrow M Neuron. 1999; 23 (this issue,): 737-746Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar measured receptor-mediated activation of inwardly rectifying potassium channels (GIRKs). Using this system, they demonstrate that morphine is only marginally less effective than DAMGO and substantially more effective than methadone in GIRK activation. Thus, the differences in the ability of morphine, DAMGO, and methadone to induce endocytosis cannot be due to differences in agonist efficacy. 8Whistler J.L. Chuang H.-H. Chu P. Jan L.Y. von Zastrow M Neuron. 1999; 23 (this issue,): 737-746Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar next probe the molecular details responsible for the differential abilities of morphine, DAMGO, and methadone to induce internalization. To accomplish this, they construct a chimeric μ/δ opioid receptor, in which the cytoplasmic C terminus of the μ receptor is replaced with the C terminus of the δ receptor. This chimeric receptor readily endocytoses in response to morphine (see figure). A critical observation is that, even though chimeric receptors are internalized after morphine exposure, morphine's relative efficacy (as measured by GIRK activation) is unaffected. They then show that the chimeric receptor can also be phosphorylated following exposure to morphine, whereas the native receptor cannot (see figure). Following receptor phosphorylation, arrestins are frequently recruited to the plasma membrane, where they facilitate endocytosis by serving as scaffolding proteins that bind to clathrin (2Ferguson S. Downey III, W.E. Colapietro A.-M. Barak L. Menard L. Caron M Science. 1996; 271: 363-366Crossref PubMed Scopus (845) Google Scholar, 3Goodman O. Krupnick J. Santini F. Gurevich V. Penn R. Gagnon A. Keen J. Benovic J Nature. 1996; 383: 447-450Crossref PubMed Scopus (1169) Google Scholar) (see figure). 8Whistler J.L. Chuang H.-H. Chu P. Jan L.Y. von Zastrow M Neuron. 1999; 23 (this issue,): 737-746Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar find that morphine fails to cause arrestin translocation from the cytosol to the plasma membrane for the native μ receptor, whereas the chimeric receptor efficiently recruits arrestin to the plasma membrane (see figure) after exposure to morphine. To determine if these results, all obtained in an artificial expression system (HEK 293 cells), are relevant to the regulation of μ receptors in neurons8Whistler J.L. Chuang H.-H. Chu P. Jan L.Y. von Zastrow M Neuron. 1999; 23 (this issue,): 737-746Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar express native and chimeric μ receptors in primary hippocampal neuronal cultures using adenovirus-mediated gene transfer. Importantly, the native receptor was unable to be internalized after morphine exposure, whereas the chimeric receptor was readily internalized. To explain these findings, the authors speculate that a measure they term RAVE might allow investigators to distinguish among ligands that have differential ultimate biological activities. They define RAVE as the relative activity (efficacy) versus the ability of a ligand to induce endocytosis. As an example, they report that methadone and etorphine, which have low propensities to induce tolerance and dependence, have low RAVE values, whereas morphine, which has a high RAVE, has a marked propensity to induce tolerance and dependence. Future studies investigating a large number of opioids (which differ in their abilities to induce analgesia, tolerance, and dependence) will be necessary to fully test this interesting notion. The report by 8Whistler J.L. Chuang H.-H. Chu P. Jan L.Y. von Zastrow M Neuron. 1999; 23 (this issue,): 737-746Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar is important to neurobiologists for several reasons. First, these findings will likely inspire a large number of addiction researchers to revisit the relevance of receptor internalization for drug dependence. More importantly, these studies will also be of interest to investigators who are identifying the general biological role(s) that endocytosis has in mediating certain molecular consequences of GPCR activation, such as MAP kinase activation (1Daaka Y. Luttrell Y.M. Ahn S. Della Rocca G.J. Ferguson S.S. Caron M.G. Lefkowitz R.J J. Biol. Chem. 1998; 273: 685-688Crossref PubMed Scopus (461) Google Scholar, 4Ignatova E.G. Belcheva M.M. Bohn L.M. Neuman M.C. Coscia C.J J. Neurosci. 1999; 19: 56-63Crossref PubMed Google Scholar). Thus, for instance, the results of 8Whistler J.L. Chuang H.-H. Chu P. Jan L.Y. von Zastrow M Neuron. 1999; 23 (this issue,): 737-746Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar suggest that if endocytosis is involved in certain biological and adaptive responses mediated by GPCRs, then individual differences in the abilities of ligands to induce endocytosis should be reflected in differential activation of downstream effectors (e.g., MAP kinase activation and induction of gene transcription). For example, as pointed out in the 8Whistler J.L. Chuang H.-H. Chu P. Jan L.Y. von Zastrow M Neuron. 1999; 23 (this issue,): 737-746Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar article, methadone and morphine differ in their abilities to induce both endocytosis and addiction. Thus, it is conceivable that other ligands which have different ultimate biological effects (i.e., psychotherapeutic drugs with different therapeutic efficacies) may have these distinct actions as a direct result of their differential abilities to produce endocytosis (9Willins D.L. Berry S.A. Alsayegh L. Backstrom J.R. Sanders-Bush E. Roth B.L Neuroscience. 1999; 91: 599-606Crossref PubMed Scopus (126) Google Scholar). Finally, the results of 8Whistler J.L. Chuang H.-H. Chu P. Jan L.Y. von Zastrow M Neuron. 1999; 23 (this issue,): 737-746Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar imply that manipulating the abilities of GPCRs to be endocytosed, either by pharmacologic or genetic approaches, could alter the ultimate biological consequences of receptor activation. That would be something about which all neuroscientists could RAVE.