Neuregulins (NRGs) and their receptors, the ErbB protein tyrosine kinases, are essential for neuronal development, but their functions in the adult CNS are unknown. We report that ErbB4 is enriched in the postsynaptic density (PSD) and associates with PSD-95. Heterologous expression of PSD-95 enhanced NRG activation of ErbB4 and MAP kinase. Conversely, inhibiting expression of PSD-95 in neurons attenuated NRG-mediated activation of MAP kinase. PSD-95 formed a ternary complex with two molecules of ErbB4, suggesting that PSD-95 facilitates ErbB4 dimerization. Finally, NRG suppressed induction of long-term potentiation in the hippocampal CA1 region without affecting basal synaptic transmission. Thus, NRG signaling may be synaptic and regulated by PSD-95. A role of NRG signaling in the adult CNS may be modulation of synaptic plasticity.

␦ and opioid receptors are homologous G proteincoupled receptors that are differentially sorted between divergent degradative and recycling membrane pathways following agonist-induced endocytosis. Whereas ␦ opioid receptors are selectively sorted to lysosomes, opioid receptors recycle rapidly to the plasma membrane by a process that has been proposed to occur via bulk membrane flow. We have observed that opioid receptors do not recycle by default and have defined a specific sequence present in the cytoplasmic tail of the cloned opioid receptor that is both necessary and sufficient for rapid recycling of internalized receptors. This sequence is completely distinct from a sequence shown previously to be required for recycling of the ␤ 2 adrenergic receptor yet is functionally interchangeable when tested in chimeric mutant receptors. These results indicate that signal-dependent recycling is a more common property of G protein-coupled receptors than previously appreciated and demonstrate that such a modular recycling signal distinguishes the regulation of homologous receptors that are naturally co-expressed.

Ubiquitination of cytoplasmic lysine residues can target G protein-coupled receptors (GPCRs) to proteasomes and has recently been shown to also be required for sorting of certain GPCRs to lysosomes following ligand-induced endocytosis. We addressed the generality of this mechanism by examining regulated proteolysis of the murine ␦ opioid receptor (DOR) expressed in human embryonic kidney 293 cells, a well characterized model system in which receptors are sorted to lysosomes. Incubation of cells in the presence of the highly specific proteasome inhibitor lactacystin did not detectably affect ligand-induced proteolysis of DOR but significantly delayed ligand-induced proteolysis of epidermal growth factor receptors. Mutation of all cytoplasmic lysine residues in DOR, creating a mutant opioid receptor that is unable to be ubiquitinated, did not detectably inhibit either ligand-induced endocytosis or proteolytic degradation of endocytosed receptors. Furthermore, the lysinemutated DOR, like its wild type counterpart, colocalized extensively with lysosomes after ligand-induced endocytosis. These results demonstrate that ubiquitination of DOR is not required either for its ligand-induced endocytosis or for postendocytic trafficking to lysosomes.Classical studies of receptor-mediated endocytosis established the principle of geometric sorting whereby membranebound receptors recycle to the plasma membrane by bulk membrane flow and require specific membrane sorting machinery to traffic to lysosomes (1-3). Although many signaling receptors are endocytosed after ligand-induced activation, there are large differences in the degree to which various receptors thereafter traffic to lysosomes. Such differences in the endocytic trafficking of receptors have important functional consequences (4 -6). In the case of G protein-coupled receptors (GPCRs), 1 events surrounding the initial endocytosis of receptors generally result in a rapid desensitization of signaling activity. Recycling of receptors back to the plasma membrane typically promotes functional resensitization, whereas sorting of internalized receptors to lysosomes leads to proteolytic down-regulation of receptors and a prolonged attenuation of signal transduction (7-9). These considerations have motivated considerable interest in elucidating mechanisms that control the sorting of specific signaling receptors in the endocytic pathway. Covalent modification of lysine residues by ubiquitin is well known to function as a signal targeting many proteins to proteasomes. Ubiquitination of cytoplasmic lysine residues has emerged more recently as a mechanism that also regulates the endocytic trafficking of various integral membrane proteins (10). Ubiquitination of lysine residues located in the carboxylterminal cytoplasmic domain of the yeast GPCR Ste2p was initially shown to promote ligand-induced endocytosis of receptors (11). More recently, ubiquitination of the cytoplasmic tail of Ste2p (12) has been shown to be essential for the subsequent sorting of endocytosed receptors to int...

Endocytic trafficking of signaling receptors to alternate intracellular pathways has been shown to lead to diverse biological consequences. In this study, we report that two neurotrophin receptors (tropomyosin-related kinase TrkA and TrkB) traverse divergent endocytic pathways after binding to their respective ligands (nerve growth factor and brain-derived neurotrophic factor). We provide evidence that TrkA receptors in neurosecretory cells and neurons predominantly recycle back to the cell surface in a ligand-dependent manner. We have identified a specific sequence in the TrkA juxtamembrane region, which is distinct from that in TrkB receptors, and is both necessary and sufficient for rapid recycling of internalized receptors. Conversely, TrkB receptors are predominantly sorted to the degradative pathway. Transplantation of the TrkA recycling sequence into TrkB receptors reroutes the TrkB receptor to the recycling pathway. Finally, we link these divergent trafficking pathways to alternate biological responses. On prolonged neurotrophin treatment, TrkA receptors produce prolonged activation of phosphatidylinositol 3-kinase/Akt signaling as well as survival responses, compared with TrkB receptors. These results indicate that TrkA receptors, which predominantly recycle in signal-dependent manner, have unique biological properties dictated by its specific endocytic trafficking itinerary.

Antisense oligonucleotide (ASO) therapeutics show tremendous promise for the treatment of previously intractable human diseases but to exert their effects on cellular RNA processing they must first cross the plasma membrane by endocytosis. The conjugation of ASOs to a receptor ligand can dramatically increase their entry into certain cells and tissues, as demonstrated by the implementation of N-acetylgalactosamine (GalNAc)-conjugated ASOs for Asialoglycoprotein Receptor (ASGR)-mediated uptake into liver hepatocytes. We compared the internalization and activity of GalNAc-conjugated ASOs and their parents in endogenous ASGR-expressing cells and were able to recapitulate hepatocyte ASO uptake and activity in cells engineered to heterologously express the receptor. We found that the minor receptor subunit, ASGR2, is not required for effective in vitro or in vivo uptake of GalNAc-conjugated ASO and that the major subunit, ASGR1, plays a small but significant role in the uptake of unconjugated phosphorothioate ASOs into hepatocytes. Moreover, our data demonstrates there is a large excess capacity of liver ASGR for the effective uptake of GalNAc–ASO conjugates, suggesting broad opportunities to exploit receptors with relatively moderate levels of expression.