Importance of Fc Receptor γ-Chain ITAM Tyrosines in Neutrophil Activation and in vivo Autoimmune Arthritis.

Activating Fcγ receptors associated with Fc receptor γ-chain (FcRγ) are critical for mediating neutrophil effector functions in immune complex-mediated autoimmune diseases. FcRγ contains ITAM tyrosines and the in vivo role of these tyrosines has not been defined in neutrophils and arthritis. In this study, the in vivo functions of FcRγ ITAM tyrosines were characterized using wild type and ITAM tyrosine mutant (Y65F/Y76F) transgenic mice crossed to an FcRγ-deficient genetic background. FcRγ-deficient neutrophils showed undetectable cell surface expression of the activating Fcγ receptor IV, defective immune complex-induced superoxide production, degranulation and spreading. Although the re-expression of both the wild type and the ITAM tyrosine mutant (Y65F/Y76F) FcRγ could restore activating Fcγ receptor expression of FcRγ-deficient neutrophils, only the wild type transgenic form could mediate Fcγ receptor-dependent effector functions. In contrast, neutrophils carrying ITAM tyrosine mutant FcRγ were unable to produce superoxide, mediate degranulation and perform active spreading. In addition, our results confirmed the protection of FcRγ-deficient mice from autoimmune arthritis. Importantly, the presence of the wild type FcRγ transgene, in contrast to the ITAM tyrosine mutant transgene, partially reversed autoimmune arthritis development. The reversing effect of the wild type transgene was even more robust when animals carried the wild type transgene in a homozygous form. Collectively, FcRγ ITAM tyrosines play a critical role in the induction of neutrophil effector responses, the initiation and progression of an autoantibody-induced experimental arthritis in vivo, indicating a signaling, rather than just a receptor stabilizing function of the molecule.

Dopamine and response selection: an Acute Phenylalanine/Tyrosine Depletion study.

The role of dopaminergic system in decision-making is well documented, and evidence suggests that it could play a significant role in response selection processes. The N-40 is a fronto-central event-related potential, generated by the supplementary motor areas (SMAs) and a physiological index of response selection processes. The aim of the present study was to determine whether infraclinical effects of dopamine depletion on response selection processes could be evidenced via alterations of the N-40. We obtained a dopamine depletion in healthy volunteers with the acute phenylalanine and tyrosine depletion (APTD) method which consists in decreasing the availability of dopamine precursors. Subjects realized a Simon task in the APTD condition and in the control condition. When the stimulus was presented on the same side as the required response, the stimulus-response association was congruent and when the stimulus was presented on the opposite side of the required response, the stimulus-response association was incongruent. The N-40 was smaller for congruent associations than for incongruent associations. Moreover, the N-40 was sensitive to the level of dopaminergic activity with a decrease in APTD condition compared to control condition. This modulation of the N-40 by dopaminergic level could not be explained by a global decrease of cerebral electrogenesis, since negativities and positivities indexing the recruitment of the primary motor cortex (anatomically adjacent to the SMA) were unaffected by APTD. The specific sensitivity of N-40 to ATPD supports the model of Keeler et al. (Neuroscience 282:156-175, 2014) according to which the dopaminergic system is involved in response selection.

Inhibition of Kv4.3 by genistein via a tyrosine phosphorylation-independent mechanism.

The effects of genistein, a protein tyrosine kinase (PTK) inhibitor, on voltage-dependent K(+) (Kv) 4.3 channel were examined using the whole cell patch-clamp techniques. Genistein inhibited Kv4.3 in a reversible, concentration-dependent manner with an IC(50) of 124.78 µM. Other PTK inhibitors (tyrphostin 23, tyrphostin 25, lavendustin A) had no effect on genistein-induced inhibition of Kv4.3. Orthovanadate, an inhibitor of protein phosphatases, did not reverse the inhibition of Kv4.3 by genistein. We also tested the effects of two inactive structural analogs: genistin and daidzein. Whereas Kv4.3 was unaffected by genistin, daidzein inhibited Kv4.3, albeit with a lower potency. Genistein did not affect the activation and inactivation kinetics of Kv4.3. Genistein-induced inhibition of Kv4.3 was voltage dependent with a steep increase over the channel opening voltage range. In the full-activation voltage range positive to +20 mV, no voltage-dependent inhibition was found. Genistein had no significant effect on steady-state activation, but shifted the voltage dependence of the steady-state inactivation of Kv4.3 in the hyperpolarizing direction in a concentration-dependent manner. The K(i) for the interaction between genistein and the inactivated state of Kv4.3, which was estimated from the concentration-dependent shift in the steady-state inactivation curve, was 1.17 µM. Under control conditions, closed-state inactivation was fitted to a single exponential function, and genistein accelerated closed-state inactivation. Genistein induced a weak use-dependent inhibition. These results suggest that genistein directly inhibits Kv4.3 by interacting with the closed-inactivated state of Kv4.3 channels. This effect is not mediated via inhibition of the PTK activity, because other types of PTK inhibitors could not prevent the inhibitory action of genistein.

The two C-terminal tyrosines stabilize occluded Na/K pump conformations containing Na or K ions.

Interactions of the three transported Na ions with the Na/K pump remain incompletely understood. Na/K pump crystal structures show that the extended C terminus of the Na,K-adenosine triphosphatase (ATPase) alpha subunit directly contacts transmembrane helices. Deletion of the last five residues (KETYY in almost all Na/K pumps) markedly lowered the apparent affinity for Na activation of pump phosphorylation from ATP, a reflection of cytoplasmic Na affinity for forming the occluded E1P(Na3) conformation. ATPase assays further suggested that C-terminal truncations also interfere with low affinity Na interactions, which are attributable to extracellular effects. Because extracellular Na ions traverse part of the membrane's electric field to reach their binding sites in the Na/K pump, their movements generate currents that can be monitored with high resolution. We report here electrical measurements to examine how Na/K pump interactions with extracellular Na ions are influenced by C-terminal truncations. We deleted the last two (YY) or five (KESYY) residues in Xenopus laevis alpha1 Na/K pumps made ouabain resistant by either of two kinds of point mutations and measured their currents as 10-mM ouabain-sensitive currents in Xenopus oocytes after silencing endogenous Xenopus Na/K pumps with 1 microM ouabain. We found the low affinity inhibitory influence of extracellular Na on outward Na/K pump current at negative voltages to be impaired in all of the C-terminally truncated pumps. Correspondingly, voltage jump-induced transient charge movements that reflect pump interactions with extracellular Na ions were strongly shifted to more negative potentials; this signals a several-fold reduction of the apparent affinity for extracellular Na in the truncated pumps. Parallel lowering of Na affinity on both sides of the membrane argues that the C-terminal contacts provide important stabilization of the occluded E1P(Na3) conformation, regardless of the route of Na ion entry into the binding pocket. Gating measurements of palytoxin-opened Na/K pump channels additionally imply that the C-terminal contacts also help stabilize pump conformations with occluded K ions.

Tyrosine-dependent and -independent actions of leptin receptor in control of energy balance and glucose homeostasis.

Leptin regulates energy balance and glucose metabolism by activation of multiple signaling cascades mediated by the long-form leptin receptor Ob-Rb. However, the whole spectrum of signaling actions through the 3 cytoplasmic tyrosines of mouse Ob-Rb remains to be completely defined in vivo. Here, we generated 2 knockin lines of mice expressing mutant leptin receptors with phenylalanine substitution for all 3 tyrosines (Y123F) or for Tyr(1138) alone (Y3F). Y123F animals developed overt obesity similar to that of Y3F animals with abrogated hypothalamic activation of STAT3 by leptin, but they exhibited more severe impairment in glucose tolerance. In striking contrast to db/db mice, however, both Y123F and Y3F mice showed attenuated adiposity with reduced hyperphagia, marked improvement in physical activity and adaptive thermogenesis, and significantly ameliorated glycemic control. Further, Y123F mice had hypothalamic neuropeptide Y/agouti-related protein expression maintained at prominently lower levels compared with db/db mice. Thus, these results provide direct physiological evidence that Ob-Rb exerts crucial metabolic actions not only through tyrosine-dependent, but also tyrosine-independent mechanisms in control of energy balance and glucose homeostasis.

Arginine stimulates intestinal cell migration through a focal adhesion kinase dependent mechanism.

BACKGROUND: L-Arginine is a nutritional supplement that may be useful for promoting intestinal repair. Arginine is metabolised by the oxidative deiminase pathway to form nitric oxide (NO) and by the arginase pathway to yield ornithine and polyamines. AIMS: To determine if arginine stimulates restitution via activation of NO synthesis and/or polyamine synthesis. METHODS: We determined the effects of arginine on cultured intestinal cell migration, NO production, polyamine levels, and activation of focal adhesion kinase, a key mediator of cell migration. RESULTS: Arginine increased the rate of cell migration in a dose dependent biphasic manner, and was additive with bovine serum concentrate (BSC). Arginine and an NO donor activated focal adhesion kinase (a tyrosine kinase which localises to cell matrix contacts and mediates beta1 integrin signalling) after wounding. Arginine stimulated cell migration was dependent on focal adhesion kinase (FAK) signalling, as demonstrated using adenovirus mediated transfection with a kinase negative mutant of FAK. Arginine stimulated migration was dependent on NO production and was blocked by NO synthase inhibitors. Arginine dependent migration required synthesis of polyamines but elevating extracellular arginine concentration above 0.4 mM did not enhance cellular polyamine levels. CONCLUSIONS: These results showed that L-arginine stimulates cell migration through NO and FAK dependent pathways and that combination therapy with arginine and BSC may enhance intestinal restitution via separate and convergent pathways.

Fos immunocytochemical studies on the neuroanatomical sites of action of acute tyrosine depletion in the rat brain.

RATIONALE: Acute depletion of brain tyrosine using a tyrosine-free amino acid mixture offers a nutritional approach to reduce central catecholamine function. Recent preclinical data suggest that tyrosine-free amino acid mixtures may have region-specific effects through targeting dopamine neurones. OBJECTIVES: Here we used fos immunocytochemistry to examine the neuroanatomical sites of action of a tyrosine-free amino acid mixture administered either alone or combined with amphetamine. METHODS: Rats (male, Sprague Dawley, 240-260 g) were administered (IP) either a tyrosine-free amino acid mixture (1 g/kg), or the same mixture supplemented with tyrosine and phenylalanine (1 g/kg). Mixtures were injected twice (1 h apart) followed 1 h later by amphetamine (2 mg/kg SC). Two hours later, cardiac perfusion was performed and brains were processed for fos immunocytochemistry. Fos positive cells were counted using computer imaging software. RESULTS: The tyrosine-free amino acid mixture alone did not alter fos expression in ten regions of the rat forebrain compared to saline controls. However, the mixture reduced the increase in fos expression evoked by amphetamine. This effect was region-specific and was greatest in caudate putamen, nucleus accumbens, bed nucleus stria terminalis and lateral habenula, and lacking in other areas including cingulate and insular cortices, lateral septum and central amygdaloid nucleus. Moreover, in most regions the effect of the tyrosine-free mixture was less after tyrosine and phenylalanine supplementation. CONCLUSIONS: In summary, a tyrosine-free amino acid mixture reduced amphetamine-induced fos expression but this effect was region-specific and included dopamine-rich regions. These data further support the idea that tyrosine depletion strategies have potential as treatments for mania and other hyperdopaminergic states.

Augmentation of endogenous dopamine production increases lung liquid clearance.

We have previously reported that dopamine increased active Na+ transport in rat lungs by upregulating the alveolar epithelial Na,K-ATPase. Here we tested whether alveolar epithelial cells produce dopamine and whether increasing endogenous dopamine production by feeding rats a 4% tyrosine diet (TSD) would increase lung liquid clearance. Alveolar Type II cells express the enzyme aromatic-L-amino acid decarboxylase (AADC) and, when incubated with the dopamine precursor, 3-hydroxy-L-tyrosine (L-dopa), produce dopamine. Rats fed TSD, a precursor of L-dopa and dopamine, had increased urinary dopamine levels, which were inhibited by benserazide, an inhibitor of AADC. Rats fed TSD for 15, 24, and 48 hours had a 26, 46, and 45% increase in lung liquid clearance, respectively, as compared with controls. Also, dopaminergic D1 receptor antagonist--but not dopaminergic D2 receptor antagonist--inhibited the TSD-mediated increase in lung liquid clearance. Alveolar Type II cells isolated from the lungs of rats after they had been fed TSD for 24 hours demonstrated increased protein abundance of Na,K-ATPase alpha1 and beta1 subunits. Basolateral membranes isolated from peripheral lung tissue of tyrosine-fed rats had increased Na,K-ATPase activity and Na,K-ATPase alpha1 subunit. These data provide the first evidence that alveolar epithelial cells produce dopamine and that increasing endogenous dopamine increases lung liquid clearance.

Subunit contributions to phosphorylation-dependent modulation of bovine rod cyclic nucleotide-gated channels.

Cyclic nucleotide-gated (CNG) channels in rod photoreceptors transduce a decrease in cGMP into hyperpolarization during the light response. Insulin-like growth factor-1 (IGF-1) increases light responses by increasing the cGMP sensitivity of CNG channels, an event mediated by a protein tyrosine phosphatase. Native rod CNG channels are heteromultimers, composed of three CNGA1 subunits and one CNGB1 subunit. Previous studies on heterologously expressed rod CNG channels show that a specific tyrosine in the CNGA1 subunit (Y498) is required for modulation by protein tyrosine phosphatases, protein tyrosine kinases and IGF-1. Here we show that the CNGB1 subunit contains a specific tyrosine (Y1097) that is important for modulation of heteromeric channels by tyrosine phosphorylation. Direct biochemical measurements demonstrate 32P-labelling of CNGA1Y498 and CNGB1Y1097. Replacement of either Y498 of CNGA1 or Y1097 of CNGB1 with phenylalanine reduces modulation, and removal of both tyrosines eliminates modulation. Unlike CNGA1, CNGB1 does not exhibit activity dependence of modulation by tyrosine phosphorylation. Hence both CNGA1 and CNGB1 subunits contribute to phosphorylation-dependent modulation of rod CNG channels, but the phosphorylation states of the two subunits are regulated in different ways.

Activating and inhibitory Ly49 receptors modulate NK cell chemotaxis to CXC chemokine ligand (CXCL) 10 and CXCL12.

NK cells can migrate into sites of inflammatory responses or malignancies in response to chemokines. Target killing by rodent NK cells is restricted by opposing signals from inhibitory and activating Ly49 receptors. The rat NK leukemic cell line RNK16 constitutively expresses functional receptors for the inflammatory chemokine CXC chemokine ligand (CXCL)10 (CXCR3) and the homeostatic chemokine CXCL12 (CXCR4). RNK-16 cells transfected with either the activating Ly49D receptor or the inhibitory Ly49A receptor were used to examine the effects of NK receptor ligation on CXCL10- and CXCL12-mediated chemotaxis. Ligation of Ly49A, either with Abs or its MHC class I ligand H2-D(d), led to a decrease in chemotactic responses to either CXCL10 or CXCL12. In contrast, Ly49D ligation with Abs or H2-D(d) led to an increase in migration toward CXCL10, but a decrease in chemotaxis toward CXCL12. Ly49-dependent effects on RNK-16 chemotaxis were not the result of surface modulation of CXCR3 or CXCR4 as demonstrated by flow cytometry. A mutation of the Src homology phosphatase-1 binding motif in Ly49A completely abrogated Ly49-dependent effects on both CXCL10 and CXCL12 chemotaxis, suggesting a role for Src homology phosphatase-1 in Ly49A/chemokine receptor cross-talk. Ly49D-transfected cells were pretreated with the Syk kinase inhibitor Piceatannol before ligation, which abrogated the previously observed changes in migration toward CXCL10 and CXCL12. Piceatannol also abrogated Ly49A-dependent inhibition of chemotaxis toward CXCL10, but not CXCL12. Collectively, these data suggest that Ly49 receptors can influence NK cell chemotaxis within sites of inflammation or tumor growth upon interaction with target cells.

Nutrition, brain function and cognitive performance.

Military interest in the effects of nutritional factors on cognitive function has stimulated considerable research on a variety of food constituents. This paper will review the research on the amino acids tryptophan and tyrosine, caffeine and carbohydrate. It will focus on research that addresses the potential utility of these compounds in military applications, particularly the acute, as opposed to chronic, effects of these substances on cognitive functions such as alertness, vigilance and resistance to stress. Caffeine, the most intensively studied food constituent, has unequivocal beneficial effects on vigilance, and in sleep deprived individuals it enhances other cognitive functions as well. Tryptophan, although it clearly has sedative-like properties, has not been extensively studied by military laboratories for use as a hypnotic, due to safety concerns. Tyrosine has been examined in animal models and human studies, and appears to prevent the substantial decline in various aspects of cognitive performance and mood associated with many kinds of acute stress. Carbohydrate supplementation appears to enhance cognitive performance in soldiers engaged in sustained, intense physical activities that expend high levels of energy.

Activated STAT4 has an essential role in Th1 differentiation and proliferation that is independent of its role in the maintenance of IL-12R beta 2 chain expression and signaling.

In this study we demonstrated that CD4(+) T cells from STAT4(-/-) mice exhibit reduced IL-12R expression and poor IL-12R signaling function. This raised the question of whether activated STAT4 participates in Th1 cell development mainly through its effects on IL-12 signaling. In a first approach to this question we determined the capacity of CD4(+) T cells from STAT4(-/-) bearing an IL-12Rbeta2 chain transgene (and thus capable of normal IL-12R expression and signaling) to undergo Th1 differentiation when stimulated by Con A and APCs. We found that such cells were still unable to exhibit IL-12-mediated IFN-gamma production. In a second approach to this question, we created Th2 cell lines (D10 cells) transfected with STAT4-expressing plasmids with various tyrosine-->phenylalanine mutations and CD4(+) T cell lines from IL-12beta2(-/-) mice infected with retroviruses expressing similarly STAT4 mutations that nevertheless express surface IL-12Rbeta2 chains. We then showed that constructs that were unable to support STAT4 tyrosine phosphorylation (in D10 cells) as a result of mutation were also incapable of supporting IL-12-induced IFN-gamma production (in IL-12Rbeta2(-/-) cells). Thus, by two complementary approaches we demonstrated that activated STAT4 has an essential downstream role in Th1 cell differentiation that is independent of its role in the support of IL-12Rbeta2 chain signaling. This implies that STAT4 is an essential element in the early events of Th1 differentiation.

Regulation of NK cell-mediated cytotoxicity by the adaptor protein 3BP2.

Stimulation of lymphocytes through multichain immune recognition receptors activates multiple signaling pathways. Adaptor proteins play an important role in integrating these pathways by their ability to simultaneously bind multiple signaling components. Recently, the 3BP2 adaptor protein has been shown to positively regulate the transcriptional activity of T cells. However, the mechanisms by which signaling components are involved in this regulation remain unclear, as does a potential role for 3BP2 in the regulation of other cellular functions. Here we describe a positive regulatory role for 3BP2 in NK cell-mediated cytotoxicity. We also identify p95(vav) and phospholipase C-gamma isoforms as binding partners of 3BP2. Our results show that tyrosine-183 of 3BP2 is specifically involved in this interaction and that this residue critically influences 3BP2-dependent function. Therefore, 3BP2 regulates NK cell-mediated cytotoxicity by mobilizing key downstream signaling effectors.

Can nutrient supplements modify brain function?

Over the past 40 y, several lines of investigation have shown that the chemistry and function of both the developing and the mature brain are influenced by diet. Examples are the effect of folate deficiency on neural tube development during early gestation, the influence of essential fatty acid deficiency during gestation and postnatal life on the development of visual function in infants, and the effects of tryptophan or tyrosine intake (alone or as a constituent of dietary protein) on the production of the brain neurotransmitters derived from them (serotonin and the catecholamines, respectively). Sometimes the functional effects are clear and the underlying biochemical mechanisms are not (as with folate and essential fatty acids); in other cases (such as the amino acids tyrosine and tryptophan), the biochemical effects are well understood, whereas the effect on brain function is not. Despite the incomplete knowledge base on the effects of such nutrients, investigators, physicians, and regulatory bodies have promoted the use of these nutrients in the treatment of disease. Typically, these nutrients have been given in doses above those believed to be required for normal health; after they have been given in pure form, unanticipated adverse effects have occasionally occurred. If this pharmacologic practice is to continue, it is important from a public safety standpoint that each nutrient be examined for potential toxicities so that appropriate purity standards can be developed and the risks weighed against the benefits when considering their use.

Systematic analysis of the role of CD19 cytoplasmic tyrosines in enhancement of activation in Daudi human B cells: clustering of phospholipase C and Vav and of Grb2 and Sos with different CD19 tyrosines.

CD19 is a coreceptor on B cells that enhances the increase in cytoplasmic calcium and ERK2 activation when coligated with the B cell Ag receptor. Constructs containing point mutations and truncations were expressed in Daudi human B lymphoblastoid cells to systematically determine the requirement for individual CD19 cytoplasmic tyrosines in these responses. Evidence for activity was found for Y330, Y360, and Y421 as well as that previously published for Y391. Precipitates formed with phosphopeptides consisting of CD19 sequences flanking these residues were used to screen for cytoplasmic proteins that mediate signaling. Phosphopeptide Y330 precipitated Grb2 and Sos, whereas phosphopeptides Y391 and Y421 both precipitated Vav and phospholipase C-gamma2. These molecules also were found associated with native CD19. In mapping studies with altered constructs, CD19 Y330 and/or Y360 were necessary for binding Grb2 and Sos. Vav associated with CD19 constitutively in unstimulated cells by a tyrosine-independent mechanism requiring the portion of CD19 encoded by exons 9-12. After B cell Ag receptor stimulation, Vav association was tyrosine-dependent, but binding was influenced by multiple residues. However, when maximally phosphorylated by pervanadate, Y391 and, to a lesser extent, Y421 were sufficient. CD19 Y391 was also both necessary and sufficient for binding phospholipase C-gamma2. Thus, different tyrosines along the CD19 cytoplasmic domain provide scaffolding for the formation of complexes of different signaling molecules.

Effect of two tyrosine mutations on the activity and regulation of the renal type II Na/Pi-cotransporter expressed in oocytes.

The rat renal type II Na/Pi-cotransporter (NaPi2), which is regulated by mechanisms involving endocytosis and lysosomal degradation, contains two sequences that show high homology with two tyrosine (Y)-based consensus motifs previously reported to be involved in such intracellular trafficking: GY402FAM matching the consensus sequence GYXXZ, and Y509RWF matching the motif YXXO. Mutations of any of these two Y nearly abolished the NaPi2 mediated 32Pi-uptake after cRNA-injection into oocytes. The mechanisms underlying these defects are however different. Mutation of the Y402 results in a lack of glycosylation and reduced surface expression of the cotransporter, that are specific for the Y402 mutation since substitution of the neighboring F403 did not have any effect. The inhibitory effect of the Y509 mutation is related to a functional inactivation of the protein expressed in the plasma membrane; mutation of the neighboring R510 also led to a decrease in the cotransporter activity. Pharmacological activation of the protein kinase C cascade by DOG induced the retrieval of both wild-type (WT) as well as Y509 cotransporters from the oocyte plasma membrane. These data suggest that the Y402 is important for the surface expression whereas Y509 for the function of the type II Na/Pi-cotransporter expressed in oocytes. Y509 seems not to be involved in the membrane retrieval of the cotransporter.

Syk tyrosine kinase and B cell antigen receptor (BCR) immunoglobulin-alpha subunit determine BCR-mediated major histocompatibility complex class II-restricted antigen presentation.

Stimulation of CD4(+) helper T lymphocytes by antigen-presenting cells requires the degradation of exogenous antigens into antigenic peptides which associate with major histocompatibility complex (MHC) class II molecules in endosomal or lysosomal compartments. B lymphocytes mediate efficient antigen presentation first by capturing soluble antigens through clonally distributed antigen receptors (BCRs), composed of membrane immunoglobulin (Ig) associated with Ig-alpha/Ig-beta heterodimers which, second, target antigens to MHC class II-containing compartments. We report that antigen internalization and antigen targeting through the BCR or its Ig-alpha-associated subunit to newly synthesized class II lead to the presentation of a large spectrum of T cell epitopes, including some cryptic T cell epitopes. To further characterize the intracellular mechanisms of BCR-mediated antigen presentation, we used two complementary experimental approaches: mutational analysis of the Ig-alpha cytoplasmic tail, and overexpression in B cells of dominant negative syk mutants. Thus, we found that the syk tyrosine kinase, an effector of the BCR signal transduction pathway, is involved in the presentation of peptide- MHC class II complexes through antigen targeting by BCR subunits.

A cytoplasmic tyrosine is essential for the basolateral localization of mutants of the human nerve growth factor receptor in Madin-Darby canine kidney cells.

Deletion of 58 internal amino acids from the C-terminal cytoplasmic domain of p75 human nerve growth factor receptor (hNGFR) changes its localization from apical to basolateral in transfected Madin-Darby Canine Kidney (MDCK) cells (Le Bivic, A., Sambuy, Y., Patzak, A., Patil, N., Chao, M., and Rodriguez-Boulan, E. (1991) J. Cell Biol. 115, 607-618). The mutant protein, PS-NGFR, also shows a dramatic increase in its ability to endocytose NGF and to recycle through basolateral endosomes. We report here the site-directed mutagenesis analysis of PS-NGFR to localize and characterize its basolateral and endocytic sorting signals. Both signals reside in the proximal part of the PS cytoplasmic tail, between positions 306 and 314. Transferring the cytoplasmic tail (19 residues) and transmembrane domain of a truncated PS mutant to the ectodomain of the placental alkaline phosphatase, an apical glypiated ectoenzyme, redirected it to the basolateral membrane and the endocytic compartments. A tyrosine at position 308, present in this short cytoplasmic segment, was mutated into phenylalanine or alanine. The resulting mutants were expressed predominantly on the apical membrane of MDCK cells. Their ability to endocytose NGF was reduced with the alanine mutant showing the stronger diminution. The PS mutant contains a short cytoplasmic sequence necessary both for basolateral targeting and endocytosis, and the requirement for tyrosine at position 308 is crucial for basolateral targeting.

Positive regulation of melanin pigmentation by two key substrates of the melanogenic pathway, L-tyrosine and L-dopa.

We describe results demonstrating the positive regulation of melanogenesis by two substrates of the melanogenic pathway. We have found that L-tyrosine and L-dihydroxyphenylalanine (L-dopa), whose metabolic fates are affected by the activity of that pathway, can also act as its regulators. In living pigment cells, tyrosinase (EC 1.14.18.1), a crucial and rate-limiting enzyme of melanogenesis, acts in subcellular organelles known as melanosomes. Melanin is laid down only in these organelles. We demonstrate that supplementing Ham's F-10 medium with additional L-tyrosine or L-dopa during the culture of amelanotic Bomirski hamster melanoma cells results in a rapid increase in melanin formation, which is not simply due to greater availability of substrate. There is a rapid increase in tyrosinase activity and a large scale synthesis of melanosomes. The effects of L-tyrosine and L-dopa are prevented by the addition of cycloheximide. The actions of L-tyrosine and L-dopa are specific in that under similar conditions D-tyrosine, D-dopa, N-acetyl-L-tyrosine, L-phenylalanine, L-tryptophan and L-valine have little or no effect. The two substrates, L-tyrosine and L-dopa, appear to act through related but distinct mechanisms. Our findings provide an example of a little-known phenomenon: regulation of a differentiated eukaryotic phenotype through positive control by substrates in the pathway.