Tyrosinase is a melanocyte-specific enzyme critical for the synthesis of melanin, a process normally restricted to a post-Golgi compartment termed the melanosome. Loss-of-function mutations in tyrosinase are the cause of oculocutaneous albinism, demonstrating the importance of the enzyme in pigmentation. In the present study, we explored the possibility that trafficking of albino tyrosinase from the endoplasmic reticulum (ER) to the Golgi apparatus and beyond is disrupted. Toward this end, we analyzed the common albino mouse mutation Tyr(C85S), the frequent human albino substitution TYR(T373K), and the temperature-sensitive tyrosinase TYR(R402Q)͞Tyr(H402A) found in humans and mice, respectively. Intracellular localization was monitored in albino melanocytes carrying the native mutation, as well as in melanocytes ectopically expressing green fluorescent protein-tagged tyrosinase. Enzymatic characterization of complex glycans and immunofluorescence colocalization with organelle-specific resident proteins established that all four mutations produced defective proteins that were retained in the ER. TYR(R402Q)͞Tyr(H402A) Golgi processing and transport to melanosomes were promoted at the permissive temperature of 32°C, but not at the nonpermissive 37°C temperature. Furthermore, evidence of protein misfolding was demonstrated by the prolonged association of tyrosinase mutants with calnexin and calreticulin, known ER chaperones that play a key role in the quality-control processes of the secretory pathway. From these results we concluded that albinism, at least in part, is an ER retention disease.calnexin ͉ protein folding ͉ quality control

Inactivation of the retinoblastoma tumor suppressor protein (pRb) has been implicated in melanoma cells, but the molecular basis for this phenotype has not yet been elucidated, and the status of additional family members (p107 and p130, together termed pocket proteins) or the consequences on downstream targets such as E2F transcription factors are not known. Because cell cycle progression is dependent on the transcriptional activity of E2F family members (E2F1–E2F6), most of them regulated by suppressive association with pocket proteins, we characterized E2F–pocket protein DNA binding activity in normal versus malignant human melanocytes. By gel shift analysis, we show that in mitogen-dependent normal melanocytes, external growth factors tightly controlled the levels of growth-promoting free E2F DNA binding activity, composed largely of E2F2 and E2F4, and the growth-suppressive E2F4–p130 complexes. In contrast, in melanoma cells, free E2F DNA binding activity (E2F2 and E2F4, to a lesser extent E2F1, E2F3, and occasionally E2F5), was constitutively maintained at high levels independently of external melanocyte mitogens. E2F1 was the only family member more abundant in the melanoma cells compared with normal melanocytes, and the approximately fivefold increase in DNA binding activity could be accounted for mostly by a similar increase in the levels of the dimerization partner DP1. The continuous high expression of cyclin D1, A2, and E, the persistent cyclin-dependent kinase 4 (CDK4) and CDK2 activities, and the presence of hyperphosphorylated forms of pRb, p107, and p130, suggest that melanoma cells acquired the capacity for autonomous growth through inactivation of all three pocket proteins and release of E2F activity, otherwise tightly regulated in normal melanocytes by external growth factors.

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OBJECTIVES-We previously demonstrated that sphingosine 1-phosphate (S1P) bimodally regulates epithelial ovarian cancer (EOC) cell invasiveness: low-concentration S1P stimulates invasion similar to lysophophatidic acid (LPA), while high-concentration S1P inhibits invasion. In this study, we investigated the mechanisms through which S1P affects EOC cell proteolysis, invasion, and adhesion in two cultured epithelial ovarian cancer cell lines. METHODS-G-proteinGi was inhibited by pertussis toxin (PTX) and GTP binding protein Rac by NSC23766. S1P conditioned media of DOV13 and OVCA429 cells were evaluated via gel zymography, fluorometric gelatinase assay, urokinase plasminogen activator (uPA) activity assay, and Western Blot for MT1-MMP. Cell invasion was analyzed in Matrigel chambers. Membrane-N-cadherin was localized via fluorescence microscopy.RESULTS-Zymography revealed pro-MMP2 in conditioned media of EOC cells regardless of treatment. Gelatinase activity was increased by low-concentration S1P. In DOV13 cells this effect was Gi and Rac dependent. In all OVCA429 and control DOV13 cells, PTX enhanced gelatinolysis, suggesting an MMP2-inhibitory pathway via Gi. MT1-MMP was decreased Gidependently by high-concentration S1P. Rac inhibition significantly counteracted low-S1P enhancement and high-S1P reduction of DOV13 invasiveness; and uPA activity in conditioned media of invading cells correlated significantly. Immunohistochemistry revealed Gi-dependent clustering of membrane-N-cadherin in DOV13 cells treated with 0.5µM S1P or 10µM LPA.CONCLUSIONS-S1P influences EOC invasion by regulating ECM-proteolysis and cell-cell attachment via MMP2, uPA, and membrane-N-cadherin. Furthermore, this study illustrates that the net effect of S1P on each of these processes reflects a complex interplay of multiple GPCR pathways involving Gi and downstream Rac.

Tyrosinase is a type I membrane glycoprotein essential for melanin synthesis. Mutations in tyrosinase lead to albinism due, at least in part, to aberrant retention of the protein in the endoplasmic reticulum and subsequent degradation by the cytosolic ubiquitin-proteasomal pathway. A similar premature degradative fate for wild type tyrosinase also occurs in amelanotic melanoma cells. To understand critical cotranslational events, the glycosylation and rate of translation of tyrosinase was studied in normal melanocytes, melanoma cells, an in vitro cell-free system, and semi-permeabilized cells. Site-directed mutagenesis revealed that all seven N-linked consensus sites are utilized in human tyrosinase. However, glycosylation at Asn-290 (Asn-GlyThr-Pro) was suppressed, particularly when translation proceeded rapidly, producing a protein doublet with six or seven N-linked core glycans. The inefficient glycosylation of Asn-290, due to the presence of a proximal Pro, was enhanced in melanoma cells possessing 2-3-fold faster (7.7-10.0 amino acids/s) protein translation rates compared with normal melanocytes (3.5 amino acids/s). Slowing the translation rate with the protein synthesis inhibitor cycloheximide increased the glycosylation efficiency in live cells and in the cell-free system. Therefore, the rate of protein translation can regulate the level of tyrosinase N-linked glycosylation, as well as other potential cotranslational maturation events.

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Chimeric transporters were constructed in which the predicted external loops of the serotonin transporter (SERT) were replaced one at a time with a corresponding sequence from the norepinephrine transporter (NET). All of the chimeric transporters were expressed at levels equal to or greater than those of wild type SERT, but the transport and binding activity of the mutants varied greatly. In particular, mutants in which the NET sequence replaced external loops 4 or 6 of SERT had transport activity 5% or less than that of wild type, and the loop 5 replacement was essentially inactive. In some of these mutants, binding of a high affinity cocaine analog was less affected than transport, suggesting that the mutation had less effect on the initial binding steps in transport than on subsequent conformational changes. The more severely affected mutants also displayed an altered response to Na ؉ . In contrast to the dramatic reduction in transport and binding, the specificity of ligand binding was essentially unchanged. Chimeric transporters did not gain affinity for dopamine, a NET substrate, or desipramine, an inhibitor, at the expense of affinity for serotonin or paroxetine, a selective SERT inhibitor. The results suggest that external loops are not the primary determinants of substrate and inhibitor binding sites. However, they are not merely passive structures connecting transmembrane segments but rather active elements responsible for maintaining the stability and conformational flexibility of the transporter. The serotonin transporter (SERT)1 is responsible for the accumulation of serotonin (5-hydroxytryptamine, 5-HT) by neurons, platelets, and other cells (1-4). It is a member of a large family of carriers that couple the uphill movement of neurotransmitters and other metabolites with the downhill flux of Na ϩ and Cl Ϫ (5). The strong homology within this family suggests that the mechanism of transport is similar and that structural elements conserved within the family perform similar functions in each transporter. A prominent feature in each of the primary sequences is the presence of 12 regions rich in hydrophobic amino acids, linked by hydrophilic regions of variable length (6, 7). The hydrophobic regions were originally modeled as membrane-spanning domains linked by alternating external and internal loops (6, 7). One of these loops, which links putative transmembrane domains 3 and 4 (TM3 and TM4), is much larger than the rest and contains consensus sites for N-linked glycosylation (8) and an intramolecular disulfide (9) in all family members.The original topological model for these NaCl-coupled transporters was challenged in studies using mutants of the ␥-aminobutyric acid (GABA) and glycine transporters (GAT-1 and GLYT-1) (10, 11). These studies suggested that the predicted first external loop (EL1) was not exposed on the cell exterior and that the first internal loop (IL1) was actually extracellular. One drawback of these studies was that the conclusions about EL1 and IL1 topology were drawn from the prope...