The cytoplasmic region of the erythropoietin receptor contains nonoverlapping positive and negative growth-regulatory domains.

The erythropoietin (EPO) receptor (EPO-R), a member of a large cytokine receptor superfamily, has a 236-amino-acid cytoplasmic region which contains no obvious tyrosine kinase or other catalytic domain. In order to delineate the linear functional domains of the cytoplasmic tail, we generated truncated mutant cDNAs which were transfected into a murine interleukin-3-dependent cell line, Ba/F3, and the EPO-dependent growth characteristics of the stable transfectants were assayed. We identified two unique domains of the cytoplasmic tail. A membrane-proximal positive signal transduction domain of less than or equal to 103 amino acids, in a region highly similar to the interleukin-2 receptor beta chain, was sufficient for EPO-mediated signal transduction. A carboxy-terminal negative-control domain, a serine-rich region of approximately 40 amino acids, increased the EPO requirement for the Ba/F3 transfectants without altering EPO-R cell surface expression, affinity for EPO, receptor oligosaccharide processing, or receptor endocytosis. Truncation of this negative-control domain allowed the Ba/F3 transfectants to grow maximally in only 1 pM EPO, 1/10 the concentration required for growth of cells expressing the wild-type EPO-R. All truncated EPO-R mutants which retained the transmembrane region of the EPO-R polypeptide bound to the gp55 envelope protein of Friend spleen focus-forming virus. Only the functional EPO-R mutants were activated by the gp55, however, suggesting that gp55- and EPO-mediated signaling occur via a similar mechanism.     

A cytosolic domain of the erythropoietin receptor contributes to endoplasmic reticulum-associated degradation.

The erythropoietin receptor (EPO-R) is the cellular target for erythropoietin (EPO), the primary hormone that mediates the proliferation of immature erythroblasts and their differentiation into mature erythrocytes. Unusual features of the EPO-R are its short half-life (t(1/2) 1-2 h), its degradation via multiple pathways and the fact that less than 1% of total cellular EPO-R molecules are found on the cell surface. The contribution of EPO-R structural determinants to the regulation of its intracellular metabolism is still unclear. The epidermal growth factor receptor (EGF-R), unlike the EPO-R, is efficiently transported to the cell surface and displays a much longer metabolic half-life. To determine which EPO-R cytosolic domains are involved in intracellular degradation, we studied chimeric receptor molecules constructed of EGF-R extracellular and transmembrane parts, linked to the full length or truncated cytosolic part of the EPO-R. The chimeras were expressed in transiently transfected COS 7 cells and stably expressed in Ba/F3 cells. Our experiments indicate that the cytosolic part of the EPO-R contains determinants that mark it for rapid degradation, in association with the endoplasmic reticulum (ER). This degradation was insensitive to brefeldin A and was inhibited by specific proteasomal inhibitors. A truncated EGF-R/EPO-R chimera containing only 50 amino acids of the EPO-R membrane-proximal cytosolic part was also rapidly degraded suggesting that these 50 amino acids are involved in receptor degradation.     

NPXY, a sequence often found in cytoplasmic tails, is required for coated pit-mediated internalization of the low density lipoprotein receptor

Rapid internalization of the cell surface low density lipoprotein (LDL) receptor requires the first 22 amino acids of the cytoplasmic domain (residues 790-811), which must include an aromatic residue at position 807. In the human LDL receptor, this position is part of a tetrameric sequence, NPVY. A similar tetramer, NPXY (where X stands for any amino acid), is conserved in LDL receptors from six species (including Xenopus laevis) and in two members of the LDL receptor gene family, human LDL receptor-related protein and rat GP330. To determine whether the NPXY sequence is necessary for coated pit-mediated internalization, we used oligonucleotide-directed mutagenesis to substitute alanines for individual amino acids in the cytoplasmic tail of the human LDL receptor. Substitution of alanine for Asn804, Pro805, or Tyr807 (but not Val806) markedly reduced internalization. Only one other amino acid in the cytoplasmic 22-mer (Phe802) was important for internalization. A review of published data revealed NPXY sequences in cytoplasmic domains of at least 10 other cell surface proteins, including tyrosine kinase-linked receptors of the epidermal growth factor and insulin receptor family, the beta-subunits of three integrin receptors, and the amyloid A4 precursor protein. This occurrence is much more frequent than would be expected by chance alone. The possibility of a conditional role for the NPXY sequence in ligand-independent internalization of these proteins is discussed.     

A transplanted NPVY sequence in the cytosolic domain of the erythropoietin receptor enhances maturation

Activation of the EPO-R [EPO (erythropoietin) receptor] by its ligand EPO promotes erythropoiesis. Low cell surface EPO-R levels are traditionally attributed to inefficient folding mediated by the receptor extracellular domain. In the present study, we addressed the role of the EPO-R intracellular domain in exit from the ER (endoplasmic reticulum) and surface expression. A fusion protein between the thermo-reversible folding mutant of VSVG (vesicular-stomatitis-virus glycoprotein) (VSVGtsO45) and the EPO-R cytosolic domain [VSVG-WT (wild-type)] displayed delayed intracellular trafficking as compared with the parental VSVGtsO45, suggesting that the EPO-R cytosolic domain can hamper ER exit. Although NPXY-based motifs were originally associated with clathrin binding and endocytosis, they may also function in other contexts of the secretory pathway. A fusion protein between VSVGtsO45 and the cytosolic portion of EPO-R containing an NPVY insert (VSVG-NPVY) displayed enhanced glycan maturation and surface expression as compared with VSVG-WT. Notably, the NPVY insert also conferred improved maturation and augmented cell surface EPO-R. Our findings highlight three major concepts: (i) the EPO-R cytosolic domain is involved in ER exit of the receptor. (ii) Sequence motifs that participate in endocytosis can also modulate transport along the secretory pathway. (iii) VSVG-fusion proteins may be employed to screen for intracellular sequences that regulate transport.     

Endocytosis of the transferrin receptor requires the cytoplasmic domain but not its phosphorylation site

The transferrin receptor (TR) mediates cellular iron uptake by bringing about the endocytosis of transferrin. We investigated whether the cytoplasmic domain of 65 N-terminal amino acids or phosphorylated sites within this domain constitute a structure that is required for TR endocytosis. To test this hypothesis, we modified the cytoplasmic serine residues or introduced a deletion of 36 amino acids by in vitro mutagenesis of a cDNA expression vector for human TR. Upon expression in transfected mouse Ltk- cells, both the wild-type and phosphorylation site mutant receptors mediated transferrin internalization, whereas the truncated receptor did not. These results provide evidence that the cytoplasmic domain, or part of it, is essential for internalization of the TR, but argue against a role for receptor phosphorylation in endocytosis.     

Class A scavenger receptor-mediated adhesion and internalization require distinct cytoplasmic domains

Class A scavenger receptors (SR-A) are transmembrane glycoproteins that mediate both ligand internalization and cell adhesion. Previous studies have identified specific amino acids in the cytoplasmic tail of SR-A that regulate receptor internalization; however, the role of cytoplasmic domains in regulating cell adhesion has not been addressed. To investigate the role of cytoplasmic domains in SR-A-mediated adhesion and to address whether SR-A-mediated adhesion and internalization require distinct cytoplasmic domains, different SR-A constructs were stably expressed in human embryonic kidney (HEK 293) cells. Deleting the entire cytoplasmic tail (SR-A Delta 1-55) greatly reduced receptor protein abundance. Retaining the six amino acids proximal to the membrane (SR-A Delta 1-49) restored receptor protein abundance. Although SR-A Delta 1-49 localized to the cell surface, cells expressing this receptor failed to internalize the ligand acetylated low density lipoprotein. Replacing the cytoplasmic tail of SR-A with that of the transferrin receptor (TfR/SR-A) resulted in retention of the chimeric receptor in the endoplasmic reticulum suggesting a specific role for the membrane-proximal amino acids in trafficking SR-A from the endoplasmic reticulum to the Golgi. Like SR-A expressing cells, cells expressing SR-A Delta 1-49 displayed increased spreading and adhesion, demonstrating that the membrane-proximal amino acids were sufficient for SR-A-mediated cell adhesion. Together, our results indicate a critical role for the membrane-proximal amino acids in SR-A trafficking and demonstrate that SR-A-mediated adhesion and internalization require distinct cytoplasmic domains.     

Ubiquitylation of leptin receptor OB-Ra regulates its clathrin-mediated endocytosis

Leptin receptors are constitutively endocytosed in a ligand-independent manner. To study their endocytosis, leptin receptors OB-Ra and OB-Rb were expressed in HeLa cells. Both receptor isoforms were ubiquitylated, internalized by clathrin-mediated endocytosis and transported to Hrs-positive endosomes after their internalization. Proteasome inhibitors inhibited OB-Ra but not OB-Rb internalization from the cell surface. OB-Ra ubiquitylation occurred on lysine residues K877 and K889 in the cytoplasmic tail, the mutation of which abolished OB-Ra internalization. Fusion of an ubiquitin molecule at the C-terminus of an OB-Ra construct defective both in ubiquitylation and endocytosis restored clathrin-dependent endocytosis of the receptor. The internalization of this constitutively mono-ubiquitylated construct was no longer sensitive to proteasome inhibitors, which inhibited OB-Ra endocytosis by blocking its ubiquitylation. Fusion of an ubiquitin molecule to a transferrin receptor deleted from its own endocytosis motif restored clathrin-mediated endocytosis. We propose that mono-ubiquitin conjugates act as internalization motifs for clathrin-dependent endocytosis of leptin receptor OB-Ra.     

Ligand-induced internalization and increased cell calcium are mediated via distinct structural elements in the carboxyl terminus of the epidermal growth factor receptor.

Signals that can mediate ligand-induced receptor internalization and calcium regulation are present in a 48-amino acid "calcium-internalization" domain in the C' terminus of the epidermal growth factor (EGF) receptor. The basis of calcium and internalization regulation signalled by this 48-amino acid sequence was analyzed using deletion and substitution mutant receptors. Cells expressing truncated receptors containing either the NH2- or COOH-terminal portion of the 48-residue domain displayed high affinity EGF-dependent endocytosis and receptor down-regulation. These endocytosis-competent EGF receptor mutants that lacked any autophosphorylation site were unable to increase the concentration of intracellular calcium. To investigate the role of self-phosphorylation in EGF-induced calcium mobilization, phenylalanine was substituted for the single autophosphorylated tyrosine residue in this region of an internalization-competent truncated receptor. The receptor-mediated calcium response was abolished, while ligand-dependent receptor internalization was unimpaired. These results demonstrate that EGF-dependent receptor endocytosis and calcium mobilization are separate events. Tyrosine self-phosphorylation is required for increased [Ca2+]i, while structural features distinct from autophosphorylation are required for receptor internalization.     

Characterization of the carboxyl-terminal domain of the rat glucose-dependent insulinotropic polypeptide (GIP) receptor. A role for serines 426 and 427 in regulating the rate of internalization.

Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone involved in the regulation of insulin secretion. In non-insulin-dependent diabetes mellitus insulin responses to GIP are blunted, possibly due to altered signal transduction or reduced receptor number. Site-directed mutagenesis was used to construct truncated GIP receptors to study the importance of the carboxyl-terminal tail (CT) in binding, signaling, and receptor internalization. Receptors truncated at amino acids 425, 418, and 405, expressed in COS-7 or CHO-K1 cells, exhibited similar binding to wild type receptors. GIP-dependent cAMP production with the 405 mutant was decreased in COS-7 cells. Maximal cAMP production in CHO-K1 cells was reduced with all truncated forms. Binding was undetectable with a receptor truncated at amino acid 400; increasing tail length by adding 5 alanines restored binding and signaling. Mutants produced by alanine scanning of residues 394-401, adjacent to transmembrane domain 7, were all functional. CT truncation by 30 or more amino acids, mutation of serines 426/427, singly or combined, or complete CT serine knockout all reduced receptor internalization rate. The majority of the GIP receptor CT is therefore not required for signaling, a minimum chain length of approximately 405 amino acids is needed for receptor expression, and serines 426 and 427 are important for regulating rate of receptor internalization.     

The SH2B1 adaptor protein associates with a proximal region of the erythropoietin receptor

Gene targeting experiments have shown that the cytokine erythropoietin (EPO), its cognate erythropoietin receptor (EPO-R), and associated Janus tyrosine kinase, JAK2, are all essential for erythropoiesis. Structural-functional and murine knock-in experiments have suggested that EPO-R Tyr-343 is important in EPO-mediated mitogenesis. Although Stat5 binds to EPO-R phosphotyrosine 343, the initial Stat5-deficient mice did not have profound erythroid abnormalities suggesting that additional Src homology 2 (SH2) domain-containing effectors may bind to EPO-R Tyr-343 and couple to downstream signaling pathways. We have utilized cloning of ligand target (COLT) screening to demonstrate that EPO-R Tyr(P)-343 and Tyr(P)-401 bind to the SH2 domain-containing adaptor protein SH2B1β. Immunoprecipitation and in vitro mixing experiments reveal that EPO-R binds to SH2B1 in an SH2 domain-dependent manner and that the sequence that confers SH2B1 binding to the EPO-R is pYXXL. Previous studies have shown that SH2B1 binds directly to JAK2, but we show that in hematopoietic cells, SH2B1β preferentially associates with the EPO-R. SH2B1 is capable of constitutive association with EPO-R, which is necessary for its optimal SH2-dependent recruitment to EPO-R-Tyr(P)-343/Tyr(P)-401. We also demonstrate that SH2B1 is responsive to EPO stimulation and becomes phosphorylated, most likely on serines/threonines, in an EPO dose- and time-dependent manner. In the absence of SH2B1, we observe enhanced activation of signaling pathways downstream of the EPO-R, indicating that SH2B1 is a negative regulator of EPO signaling.     

Role of the human transferrin receptor cytoplasmic domain in endocytosis: localization of a specific signal sequence for internalization

Wild-type and mutant human transferrin receptors have been expressed in chicken embryo fibroblasts using a helper-independent retroviral vector. The internalization of mutant human transferrin receptors, in which all but four of the 61 amino acids of the cytoplasmic domain had been deleted, was greatly impaired. However, when expressed at high levels, such "tailless" mutant receptors could provide chicken embryo fibroblasts with sufficient iron from diferric human transferrin to support a normal rate of growth. As the rate of recycling of the mutant receptors was not significantly different from wild-type receptors, an estimate of relative internalization rates could be obtained from the distribution of receptors inside the cell and on the cell surface under steady-state conditions. This analysis and the results of iron uptake studies both indicate that the efficiency of internalization of tailless mutant receptors is approximately 10% that of wild-type receptors. Further studies of a series of mutant receptors with different regions of the cytoplasmic domain deleted suggested that residues within a 10-amino acid region (amino acids 19-28) of the human transferrin receptor cytoplasmic domain are required for efficient endocytosis. Insertion of this region into the cytoplasmic domain of the tailless mutant receptors restored high efficiency endocytosis. The only tyrosine residue (Tyr 20) in the cytoplasmic domain of the human transferrin receptor is found within this 10-amino acid region. A mutant receptor containing glycine instead of tyrosine at position 20 was estimated to be approximately 20% as active as the wild-type receptor. We conclude that the cytoplasmic domain of the transferrin receptor contains a specific signal sequence located within amino acid residues 19-28 that determines high efficiency endocytosis. Further, Tyr 20 is an important element of that sequence.     

Ligand-induced internalization and recycling of the human neuropeptide Y2 receptor is regulated by its carboxyl-terminal tail

Agonist-induced internalization of G protein-coupled receptors plays an important role in signal regulation. The underlying mechanisms of the internalization of the human neuropeptide Y(2) receptor (hY(2)R), as well as its desensitization, endocytosis, and resensitization are mainly unknown. In the present study we have investigated the role of carboxyl-terminal (C-terminal) Ser/Thr residues and acidic amino acids in regulating receptor internalization, arrestin interaction, and recycling by fluorescence microscopy, cell surface enzyme-linked immunosorbent assay, and bioluminescence resonance energy transfer in several cell lines. Strikingly, C-terminal truncation mutants revealed two different internalization motifs. Whereas a distal motif (373)DSXTEXT(379) was found to be the primary regulatory internalization sequence acting in concert with arrestin-3, the proximal motif (347)DXXXSEXSXT(356) promoted ligand-induced internalization in an arrestin-3-independent manner. Moreover, we identified a regulatory sequence located between these internalization motifs ((357)FKAKKNLEVRKN(368)), which serves as an inhibitory element. We found that hY(2)R recycling is also governed by structural determinants within the proximal internalization motif. In conclusion, these results indicate that the hY(2)R C terminus is involved in multiple molecular events that regulate internalization, interaction with arrestin-3, and receptor resensitization. Our findings provide novel insights into complex mechanisms of controlled internalization of hY(2)R, which is likely applicable to other GPCRs.     

Cytosolic lysine residues enhance anterograde transport and activation of the erythropoietin receptor

Lysine residues are key residues in many cellular processes, in part due to their ability to accept a wide variety of post-translational modifications. In the present study, we identify the EPO-R [EPO (erythropoietin) receptor] cytosolic lysine residues as enhancers of receptor function. EPO-R drives survival, proliferation and differentiation of erythroid progenitor cells via binding of its ligand EPO. We mutated the five EPO-R cytosolic lysine residues to arginine residues (5KR EPO-R), eliminating putative lysine-dependent modifications. Overexpressed 5KR EPO-R displayed impaired ubiquitination and improved stability compared with wt (wild-type) EPO-R. Unexpectedly, fusion proteins consisting of VSVGtsO45 (vesicular stomatitis virus glycoprotein temperature-sensitive folding mutant) with wt or 5KR EPO-R cytosolic domains demonstrated delayed glycan maturation kinetics upon substitution of the lysine residues. Moreover, VSVG-wt EPO-R, but not VSVG-5KR EPO-R, displayed endoplasmic reticulum-associated ubiquitination. Despite similar cell-surface EPO-binding levels of both receptors and the lack of EPO-induced ubiquitination by 5KR EPO-R, the lysine-less mutant produced weaker receptor activation and signalling than the wt receptor. We thus propose that EPO-R cytosolic lysine residues enhance receptor function, most probably through ubiquitination and/or other post-translational modifications.     

Role of the carboxyl-terminal region, di-leucine motif and cysteine residues in signalling and internalization of vasopressin V1a receptor.

The structural requirements for internalization and signalling of the vasopressin V1a receptor were investigated in stably transfected HEK-293 cells. Removal of the 51 C-terminal amino acids did not affect vasopressin binding, calcium signalling, heterologous desensitization or internalization of the receptor. Deletion of 14 additional amino acids reduced vasopressin-dependent calcium increase and impaired receptor internalization. Substitution of cysteines 371-372 did not affect intracellular signalling, but decreased endocytosis by 26%. Substitution of the 361-362 leucine by alanine residues reduced by 56% V1a receptor sequestration without affecting calcium signalling. These results indicate that di-cysteine and mostly di-leucine motifs present in the C-terminal region of the V1a receptor are involved in its internalization.     

Mutations in the WSAWSE and cytosolic domains of the erythropoietin receptor affect signal transduction and ligand binding and internalization.

The terminal development of erythroid progenitor cells is promoted in part through the interaction of erythropoietin (EPO) with its cell surface receptor. This receptor and a growing family of related cytokine receptors share homologous extracellular features, including a well-conserved WSXWS motif. To explore the functional significance of this motif in the murine EPO receptor, five WSAWSE mutants were prepared and their signal-transducing, ligand binding, and endocytotic properties were compared. EPO receptors mutated at tryptophan residues (W-232, W-235----G; W-235----G; W-235----F) failed to mediate EPO-induced growth or pp100 phosphorylation, while S-236----T and E-237----K mutants exhibited partial to full activity (50 to 100% of wild-type growth and induced phosphorylation). Ligand affinity was reduced for mutant receptors (two- to fivefold), yet expression at the cell surface for all receptors was nearly equivalent. Also, the ability of mutated receptors to internalize ligand was either markedly reduced or abolished (W-235----F), indicating a role for the WSAWSE region in hormone internalization. Interestingly, receptor forms lacking 97% of the cytosolic domain (no signal-transducing capacity; binding affinity reduced two- to threefold) internalized EPO efficiently. This and all WSAWSE receptor forms studied also mediated specific cross-linking of 125I-EPO to three accessory membrane proteins (M(r)s, 120,000, 105,000, and 93,000). These findings suggest that the WSAWSE domain of the EPO receptor is important for EPO-induced signal transduction and ligand internalization. In contrast, although the cytosolic domain is required for growth signaling, it appears nonessential for efficient endocytosis.     

Amino acids in the cytoplasmic C terminus of the parathyroid Ca2+-sensing receptor mediate efficient cell-surface expression and phospholipase C activation

The C-terminal tail of the calcium receptor (CaR) regulates the affinity of the receptor for ligand, desensitization, and membrane localization. To determine the role of specific amino acids in the bovine parathyroid CaR in mediating signal transduction and cell-surface expression, we transfected truncated and mutated CaR cDNAs into HEK-293 cells. The ability of high extracellular [Ca(2+)] ([Ca(2+)](o)) to increase total inositol phosphate (InsP) production, an index of phospholipase C (PLC) activation, was determined. Receptor expression was assessed by immunoblotting and immunocytochemistry. In cells transiently or stably expressing receptors with the C-terminal tail truncated after residue 895 (CaR-(1-895)) or 929 (CaR-(1-929)), raising [Ca(2+)](o) increased InsPs to levels comparable with those of cells expressing wild-type CaRs. There were no PLC responses to high [Ca(2+)](o) (up to 30 mm) in cells expressing CaRs with C-terminal tails of only 3 residues (CaR-(1-866)), even though these receptors were expressed in the membrane. We scanned the residues between Ser(866) and Val(895) using tandem-Ala and single-site mutagenesis. Two point mutants (His(880) --> Ala and Phe(882) --> Ala CaR) showed 50-70% reductions in high [Ca(2+)](o)-induced InsP production. The levels of expression and glycosylation of these mutants were comparable with wild-type CaRs, but both receptors were profoundly retained in intracellular organelles and co-localized with the endoplasmic reticulum marker BiP. This suggested that the signaling defects of these receptors were likely because of defective trafficking of receptors to the cell surface. Modeling of the C-terminal domain of the CaR indicated that His(880) and Phe(882) are situated in a putative alpha-helical structure of 15 amino acids between residues 877 and 891 in the C-terminal tail. Our studies support the idea that specific amino acids, and possibly a unique secondary structure in the C-terminal tail, are required for the efficient targeting of the CaR to the cell surface required for PLC activation.     

Functional study of the Saccharomyces cerevisiae Nha1p C-terminus

Saccharomyces cerevisiae cells possess an alkali metal cation antiporter encoded by the NHA1 gene. Nha1p is unique in the family of yeast Na+/H+ antiporters on account of its broad substrate specificity (Na+, Li+, K+) and its long C-terminus (56% of the whole protein). In order to study the role of the C-terminus in Nha1p function, we constructed a series of 13 truncated NHA1 versions ranging from the complete one (2958 nucleotides, 985 amino acids) down to the shortest version (1416 nucleotides, 472 amino acids), with only 41 amino acid residues after the last putative transmembrane domain. Truncated NHA1 versions were expressed in an S. cerevisiae alkali metal cation-sensitive strain (B31; ena1-4Delta nha1Delta). We found that the entire Nha1p C-terminus domain is not necessary for either the proper localization of the antiporter in the plasma membrane or the transport of all four substrates (we identified rubidium as the fourth Nha1p substrate). Partial truncation of the C-terminus of about 70 terminal amino acids improves the tolerance of cells to Na+, Li+ and Rb+ compared with cells expressing the complete Nha1p. The presence of the neighbouring part of the C-terminus (amino acids 883-928), rich in aspartate and glutamate residues, is necessary for the maintenance of maximum Nha1p activity towards sodium and lithium. In the case of potassium, the participation of the long C-terminus in the regulation of intracellular potassium content is demonstrated. We also present evidence that the Nha1p C-terminus is involved in the cell response to sudden changes in environmental osmolarity.     

Requirements for Recruitment of a G Protein-coupled Receptor to Clathrin-coated Pits in Budding Yeast

Endocytic internalization of G protein-coupled receptors (GPCRs) plays a critical role in down-regulation of GPCR signaling. The yeast mating pheromone receptor Ste2p has been used as a model to investigate mechanisms of signal transduction, modification, and endocytic internalization of GPCRs. We previously used a fluorescently labeled mating pheromone derivative to reveal unappreciated molecular and spatiotemporal features of GPCR endocytosis in budding yeast. Here, we identify recruitment of Ste2p to preexisting clathrin-coated pits (CCPs) as a key step regulated by receptor phosphorylation and subsequent ubiquitination upon ligand binding. The yeast casein kinase I homologue Yck2p directly phosphorylates six serine residues located in the C-terminal tail of Ste2p, and mutation of these serine residues to alanine significantly decreased recruitment of Ste2p to CCPs. We also found that the clathrin adaptors Ent1p, Ent2p, and Ede1p work cooperatively to recruit ubiquitinated Ste2p to CCPs. In addition, ubiquitination has a role in ligand-independent constitutive recruitment of Ste2p to CCPs, although this process is much slower than ligand-induced recruitment. These results suggest that ubiquitination of Ste2p is indispensable for recruiting Ste2p to CCPs in both ligand-dependent and ligand-independent endocytosis.     

The extracellular N terminus of the endothelin B (ETB) receptor is cleaved by a metalloprotease in an agonist-dependent process

The extracellular N terminus of the endothelin B (ET(B)) receptor is susceptible to limited proteolysis (cleavage at R64 downward arrow S65), but the regulation and the functional consequences of the proteolysis remain elusive. We analyzed the ET(B) receptor or an ET(B)-GFP fusion protein stably or transiently expressed in HEK293 cells. After incubation of cells at 4 degrees C, only the full-length ET(B) receptor was detected at the cell surface. However, when cells were incubated at 37 degrees C, N-terminal cleavage was observed, provided endothelin 1 was present during the incubation. Cleavage was not inhibited by internalization inhibitors (sucrose, phenylarsine oxide). However, in cells incubated with both internalization inhibitors and metalloprotease inhibitors (batimastat, inhibitor of TNFalpha-convertase) or metal chelators (EDTA, phenanthroline), the cleavage was blocked, indicating that metalloproteases cleave the agonist-occupied ET(B) receptor at the cell surface. Functional analysis of a mutant ET(B) receptor lacking the first 64 amino acids ([Delta2-64]ET(B) receptor) revealed normal functional properties, but a 15-fold reduced cell surface expression. The results suggest a role of the N-terminal proteolysis in the regulation of cell surface expression of the ET(B) receptor. This is the first example of a multispanning membrane protein, which is cleaved by a metalloprotease, but retains its functional activity and overall structure.