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F J Lee

Publications and source records attributed to F J Lee.

At least 19 recordsLinked to original sources

Does learning a complex task have to be complex? A study in learning decomposition.

Many theories of skill acquisition have had considerable success in addressing the fine details of learning in relatively simple tasks, but can they scale up to complex tasks that are more typical of human learning in the real world? Some theories argue for scalability by making the implicit assumption that complex tasks consist of many smaller parts, which are learned according to basic learning principles. Surprisingly, there has been rather sparse empirical testing of this crucial assumption. In this article, we examine this assumption directly by decomposing the learning in the Kanfer-Ackerman Air-Traffic Controller Task (Ackerman, 1988) from the learning at the global level all the way down to the learning at the keystroke level. First, we reanalyze the data from Ackerman (1988) and show that the learning in this complex task does indeed reflect the learning of smaller parts at the keystroke level. Second, in a follow-up eye-tracking experiment, we show that a large portion of the learning at the keystroke level reflects the learning even at a lower, i.e., attentional level.

Attention↗

Direct binding and functional coupling of alpha-synuclein to the dopamine transporters accelerate dopamine-induced apoptosis.

Mutations in alpha-synuclein, a protein highly enriched in presynaptic terminals, have been implicated in the expression of familial forms of Parkinson's disease (PD) whereas native alpha-synuclein is a major component of intraneuronal inclusion bodies characteristic of PD and other neurodegenerative disorders. Although overexpression of human alpha-synuclein induces dopaminergic nerve terminal degeneration, the molecular mechanism by which alpha-synuclein contributes to the degeneration of these pathways remains enigmatic. We report here that alpha-synuclein complexes with the presynaptic human dopamine transporter (hDAT) in both neurons and cotransfected cells through the direct binding of the non-A beta amyloid component of alpha-synuclein to the carboxyl-terminal tail of the hDAT. alpha-Synuclein--hDAT complex formation facilitates the membrane clustering of the DAT, thereby accelerating cellular dopamine uptake and dopamine-induced cellular apoptosis. Since the selective vulnerability of dopaminergic neurons in PD has been ascribed in part to oxidative stress as a result of the cellular overaccumulation of dopamine or dopamine-like molecules by the presynaptic DAT, these data provide mechanistic insight into the mode by which the activity of these two proteins may give rise to this process.

Apoptosis↗

ARL4, an ARF-like protein that is developmentally regulated and localized to nuclei and nucleoli.

ADP-ribosylation factors (ARFs) are highly conserved approximately 20-kDa guanine nucleotide-binding proteins that participate in both exocytic and endocytic vesicular transport pathways via mechanisms that are only partially understood. Although several ARF-like proteins (ARLs) are known, their biological functions remain unclear. To characterize its molecular properties, we cloned mouse and human ARL4 (mARL4 and hARL4) cDNA. The appearance of mouse ARL4 mRNA during embryonic development coincided temporally with the sequential formation of somites and the establishment of brain compartmentation. Using ARL4-specific antibody for immunofluorescence microscopy, we observed that endogenous mARL4 in cultured Sertoli and neuroblastoma cells was mainly concentrated in nuclei. When expressed in COS7 cells, ARL4-T34N mutant, predicted to exist with GDP bound, was concentrated in nucleoli. Yeast two-hybrid screening and in vitro protein-interaction assays showed that hARL4 interacted with importin-alpha through its C-terminal NLS region and that the interaction was not nucleotide-dependent. Like ARL2 and -3, recombinant hARL4 did not enhance cholera toxin-catalyzed auto-ADP-ribosylation. Its binding of guanosine 5'-O-(thiotriphosphate) was modified by phospholipid and detergent, and the N terminus of hARL4, like that of ARF, was myristoylated. Our findings suggest that ARL4, with its distinctive nuclear/nucleolar localization and pattern of developmental expression, may play a unique role(s) in neurogenesis and somitogenesis during embryonic development and in the early stages of spermatogenesis in adults.

ADP-Ribosylation Factors↗

A cognate dopamine transporter-like activity endogenously expressed in a COS-7 kidney-derived cell line.

The activity of the dopamine transporter is an important mechanism for the maintenance of normal dopaminergic homeostasis by rapidly removing dopamine from the synaptic cleft. In kidney-derived COS-7, COS-1 and HEK-293 but not in other mammalian cell lines (CHO, Y1, Ltk-), we have characterized a putative functional dopamine transporter displaying a high affinity (Km approximately 250 nM) and a low capacity (approximately 0.1 pmol/10(5) cells/min) for [3H]dopamine uptake. Uptake displayed a pharmacological profile clearly indicative of the neuronal dopamine transporter. Estimated Ki values of numerous substrates and inhibitors for the COS-dopamine transporter and the cloned human neuronal transporter (human dopamine transporter) correlate well with the exception of a few notable compounds, including the endogenous neurotransmitter dopamine, the dopamine transporter inhibitor GBR 12,909 and the dopaminergic agonist apomorphine. As with native neuronal and cloned dopamine transporters, the uptake velocity was sodium-sensitive and reduced by phorbol ester pre-treatment. Two mRNA species of 3.8 and 4.0 kb in COS-7 cells were revealed by Northern blot analysis similar in size to that seen in native neuronal tissue. A reverse-transcribed PCR analysis confirmed the existence of a processed dopamine transporter. However, no immunoreactive proteins of expected dopamine transporter molecular size or [3H]WIN 35,428 binding activity were detected. A partial cDNA of 1.3 kb, isolated from a COS-1 cDNA library and encoding transmembrane domains 1-6, displayed a deduced amino acid sequence homology of approximately 96% to the human dopamine transporter. Taken together, the data suggest the existence of a non-neuronal endogenous high affinity dopamine uptake system sharing strong functional and molecular homology to that of the cloned neuronal dopamine transporter.

Animals↗

Structural elements of ADP-ribosylation factor 1 required for functional interaction with cytohesin-1.

ADP-ribosylation factor 1 (ARF1) is a 20-kDa guanine nucleotide-binding protein involved in vesicular trafficking. Conversion of inactive ARF-GDP to active ARF-GTP is catalyzed by guanine nucleotide exchange proteins such as cytohesin-1. Cytohesin-1 and its Sec7 domain (C-1Sec7) exhibit guanine nucleotide exchange protein activity with ARF1 but not ARF-like protein 1 (ARL1), which is 57% identical in amino acid sequence. With chimeric proteins composed of ARF1 (F) and ARL1 (L) sequences we identified three structural elements responsible for this specificity. Cytohesin-1 increased [35S]guanosine 5'-(gamma-thio)triphosphate binding to L28/F (first 28 residues of L, remainder F) and to a much lesser extent F139/L, and mut13F139/L (F139/L with random sequence in the first 13 positions) but not Delta13ARF1 that lacks the first 13 amino acids; therefore, a nonspecific ARF N terminus was required for cytohesin-1 action. The N terminus was not, however, required for that of C-1Sec7. Both C-1Sec7 and cytohesin-1 effectively released guanosine 5'-(gamma-thio)triphosphate from ARF1, but only C-1Sec7 displaced the nonhydrolyzable GTP analog bound to mut13F139/L, again indicating that structure in addition to the Sec7 domain is involved in cytohesin-1 interaction. Some element(s) of the C-terminal region is also involved, because replacement of the last 42 amino acids with ARL sequence in F139L decreased markedly the interaction with cytohesin-1. Participation of both termini is consistent with the crystallographic structure of ARF in which the two terminal alpha-helices are in close proximity. ARF1 residues 28-50 are also important in the interaction with cytohesin-1; replacement of Lys-38 with Gln, the corresponding residue in ARL1, abolished the ability to serve as substrate for cytohesin-1 or C-1Sec7. These studies have defined multiple structural elements in ARF1, including switch 1 and the N and C termini, that participate in functional interactions with cytohesin-1 (or its catalytic domain C-1Sec7), which were not apparent from crystallographic analysis.

ADP-Ribosylation Factor 1↗

Characterization of a novel ADP-ribosylation factor-like protein (yARL3) in Saccharomyces cerevisiae.

ADP-ribosylation factors (ARFs) are highly conserved, approximately 20-kDa guanine nucleotide-binding proteins that enhance the ADP-ribosyltransferase activity of cholera toxin and have an important role in vesicular transport. Several cDNAs for ARF-like proteins (ARLs) have been cloned from human, Drosophila, rat, and yeast, although the biological function(s) of ARLs is unknown. We have identified a yeast gene (yARL3) encoding a protein that is structurally related (>43% identical) to the mammalian ARF-like protein ARP. Biochemical studies of purified recombinant yARL3 protein revealed properties similar to those of ARF and ARL proteins, including the ability to bind and hydrolyze GTP. Like other ARLs, recombinant yARL3 did not stimulate cholera toxin-catalyzed auto-ADP-ribosylation. Anti-yARL3 antibodies did not cross-react with yARFs or yARL1. yARL3 was not essential for cell viability, but disruption of yARL3 resulted in cold-sensitive cell growth. At the nonpermissive temperature, processing of alkaline phosphatase and carboxypeptidase Y in arl3 mutant was slowed. yARL3 might be required for protein transport from endoplasmic reticulum to Golgi or from Golgi to vacuole at nonpermissive temperatures. On subcellular fractionation, unlike its mammalian homologue ARP, yARL3 was detected in the soluble fraction but not in the plasma membrane. Indirect immunofluorescence analysis revealed that yARL3 when overexpressed was associated in part with the endoplasmic reticulum-nuclear envelope. Thus, the structural and functional characteristics of yARL3 indicate that it may have a unique role(s) in vesicular trafficking.

ADP-Ribosylation Factors↗

Identification and subcellular localization of a novel Cu,Zn superoxide dismutase of Mycobacterium tuberculosis.

Periplasmic copper, zinc superoxide dismutases (Cu,ZnSOD) of several Gram-negative pathogens have been shown to play an important role in protection against exogenous superoxide radicals and in determining virulence of the pathogens. Here we report the cloning and characterization of the sodC gene, encoding Cu,ZnSOD, from the Gram-positive Mycobacterium tuberculosis. The predicted protein sequence contains 240 amino acids with a putative signal peptide at the N-terminus and shows approximately 25% identity to other bacterial sodC. Recombinant proteins of a full-length sodC and a truncated form lacking the putative signal peptide were overexpressed in Escherichia coli and affinity purified. Renatured recombinant M. tuberculosis sodC protein possessed characteristics of a Cu,ZnSOD. Immunoblotting with an antiserum against the recombinant M. tuberculosis Cu,ZnSOD allowed detection of a single polypeptide in the lysate of M. tuberculosis. This polypeptide has a similar size as the recombinant protein without the putative signal peptide indicating that the endogenous Cu,ZnSOD in M. tuberculosis might be processed and secreted. Furthermore, immunogold electron microscopic image showed that Cu,ZnSOD is located in the periphery of M. tuberculosis. The enzymatic activity and subcellular localization of this novel Cu,ZnSOD suggest that it may play a role in determining virulence of M. tuberculosis.

Amino Acid Sequence↗

Phospholipid- and GTP-dependent activation of cholera toxin and phospholipase D by human ADP-ribosylation factor-like protein 1 (HARL1).

ADP-ribosylation factors (ARFs), 20-kDa guanine nucleotide-binding proteins named for their ability to activate cholera toxin (CT) ADP-ribosyltransferase activity, have a critical role in vesicular transport and activate a phospholipase D (PLD) isoform. Although ARF-like (ARL) proteins are very similar in sequence to ARFs, they were initially believed not to activate CT or PLD. mRNA for human ARL1 (hARL1), which is 57% identical in amino acid sequence to hARF1, is present in all tissues, with the highest amounts in kidney and pancreas and barely detectable amounts in brain. Relative amounts of hARL1 protein were similar to mRNA levels. Purified hARL1 (rARL1) synthesized in Escherichia coli had less activity toward PLD than did rARF1, although PLD activation by both proteins was guanosine guanosine 5'-(gamma-thio)triphosphate (GTPgammaS)-dependent. ARL1 stimulation of CT-catalyzed ADP-ribosylation was considerably less than that by rARF1 and was phospholipid dependent. GTPgammaS-binding by rARL1 was also phospholipid- and detergent-dependent, and in assays containing phosphatidylserine, was greater than that by rARF1. In vitro, the activities of rARL1 and rARF1 are similar. Rather than being a member of a separate subfamily, hARL1, which activates PLD and CT in a phospholipiddependent manner, appears to be part of a continuum of ARF family proteins.

ADP-Ribosylation Factors↗

Protein kinase-mediated bidirectional trafficking and functional regulation of the human dopamine transporter.

Modification of the transport velocity of both the native neuronal and cloned presynaptic dopamine transporter (DAT) has been reported following activation/inhibition of second messenger system pathways. In order to identify the mechanism by which the functional activity of human DAT (hDAT) is regulated, we assessed the [3H]dopamine uptake kinetics, [3H] CFT binding characteristics, and, via immunofluorescent confocal microscopy, the cellular localization profiles of the hDAT expressed in both Sf9 and COS-7 cells following modulation of protein kinase C (PKC)- and protein kinase A (PKA)-dependent pathways. As with both native neuronal and cloned DATs, acute exposure of hDAT expressing Sf9 cells to the PKC activator PMA (1 microM), but not alphaPDD, reduced the Vmax (approximately 1 pmol/min/10(5) cells) for [3H]DA uptake by approximately 40%, an effect which was blocked by the protein kinase inhibitor staurosporine. Pretreatment of cells with staurosporine (500 nM) alone, however, increased [3H]DA uptake velocity by approximately 30%, an effect mimicked by the potent PKA inhibitor Rp-cAMPS. Activation of PKA-dependent pathways with Sp-cAMPS did not significantly modify DA uptake. Neither the Km of [3H]DA uptake (approximately 200 nM) nor the affinity of various substrates and transport inhibitors was altered by either PMA or staurosporine treatment. Despite changes in functional dopamine uptake velocity by PKC/PKA-dependent mechanisms, the estimated density of hDAT as indexed by whole-cell [3H] CFT binding was unchanged. Immunofluorescent confocal microscopy demonstrated that the observed functional consequence of PKC activation on [3H]DA uptake is associated with the rapid sequestration/internalization of hDAT protein from the cell surface, while the increase in DA uptake following PKC/PKA inhibition is the result of the recruitment of internalized or intracellular transporters to the plasma membrane. Identical rapid translocation patterns were observed in similarly treated COS-7 cells transiently expressing hDAT. These data suggest that the differential regulation of DAT transport capacity by both PKC- and PKA-dependent pathways are not a result of modifications in DAT catalytic activity. Moreover, the rapid shuttling of DATs between the plasma membrane and intracellular compartments provides an efficient means by which native DAT function may be regulated by second messenger systems, possibly following activation of presynaptic dopaminergic receptors, and suggests a role for cytoskeletal components in the dynamic regulation of DAT function.

Animals↗

Characterization of an ADP-ribosylation factor-like 1 protein in Saccharomyces cerevisiae.

ADP-ribosylation factors (ARFs) are highly conserved approximately 20-kDa guanine nucleotide-binding proteins that enhance the ADP-ribosyltransferase activity of cholera toxin and are believed to participate in vesicular transport in both exocytic and endocytic pathways. Several ARF-like proteins (ARLs) have been cloned from Drosophila, rat, and human; however, the biological functions of ARLs are unknown. We have identified a yeast gene (ARL1) encoding a protein that is structurally related (>60% identical) to human, rat, and Drosophila ARL1. Biochemical analyses of purified recombinant yeast ARL1 (yARL1) protein revealed properties similar to those ARF and ARL1 proteins, including the ability to bind and hydrolyze GTP. Like other ARLs, recombinant yARL1 protein did not stimulate cholera toxin-catalyzed auto-ADP-ribosylation. yARL1 was not recognized by antibodies against mammalian ARLs or yeast ARFs. Anti-yARL1 antibodies did not cross-react with yeast ARFs, but did react with human ARLs. On subcellular fractionation, yARL1, similar to yARF1, was localized to the soluble fraction. The amino terminus of yARL1, like that of ARF, was myristoylated. Unlike Drosophila Arl1, yeast ARL1 was not essential for cell viability. Like rat ARL1, yARL1 might be associated in part with the Golgi complex. However, yARL1 was not required for endoplasmic reticulum-to-Golgi protein transport, and it may offer an opportunity to define an ARL function in another kind of vesicular trafficking, such as the regulated secretory pathway.

ADP-Ribosylation Factors↗

A N(alpha)-acetyltransferase selectively transfers an acetyl group to NH2-terminal methionine residues: purification and partial characterization.

Methionine N(alpha)-acetyltransferase (M-N(alpha)AT), which selectively catalyzes the transfer of an acetyl group from acetyl coenzyme A to the alpha-NH2 group of methionine residues in proteins and peptides, was isolated from Saccharomyces cerevisiae. The enzyme was purified 22000-fold to apparent homogeneity by successive purification steps using DEAE-Sepharose, DE-52 cellulose, CM-52 cellulose, Affi-Gel Blue gel and hydroxyapatite. The Mr of the native enzyme was estimated to be 70000 +/- 5000 by gel filtration chromatography. The enzyme has a pI near 8.3 as determined by chromatofocusing on Mono P. The enzyme catalyzed the transfer of an acetyl group to a synthetic peptide mimicking the first 24 residues of yeast proteinase A inhibitor 3 (Met-Asn-Thr...) and 3 of its 19 penultimately substituted analogues ([Asp2], [Glu2], and [Gln2]). Based on the estimated molecular weight and amino-acid sequence, The enzyme is different from two other recently identified methionine N(alpha)-acetyltransferases, NAT2 (Kulkarni, M.S. and Sherman, F. (1994) J. Biol. Chem. 269, 13141-13147) and MAK3 (Tercero, J.C. and Wickner, R.B. (1992) J. Biol. Chem. 267, 20277-20281). Among these three enzymes, M-N(alpha)AT and NAT2 have similar substrate specificity, however, only purified M-N(alpha)AT, but not recombinant NAT2 gene product, can catalyze the transfer of acetyl group to NH2-terminal methionine residues. The availability of this methionine N(alpha)-acetyltransferase will advance the understanding of protein co-translational processing.

Acetylation↗

Acetyl-CoA hydrolase involved in acetate utilization in Saccharomyces cerevisiae.

Acetyl-CoA hydrolase, catalyzing the hydrolysis of acetyl-CoA, is presumably involved in regulating the intracellular acetyl-CoA or CoASH pools. The yeast enzyme is encoded by ACHl (acetyl-CoA hydrolase) and the expression of ACH1 is repressed by glucose (Lee, F.-J.S., Lin, L.-W. and Smith, J.A. (1990) J. Biol. Chem. 265, 7413-7418). In order to study the biological function of the acetyl-CoA hydrolase, a null mutation (achl-1) was created by gene replacement. The mutation, while not lethal, slows down acetate utilization. In comparison to wild-type, homozygote achl-l diploids, the onset of sporulation was delayed. When measuring the levels of ACH1 mRNA and acetyl-CoA hydrolase activity, we demonstrated that ACHl was highly expressed during sporulation process. These results indicated that acetyl-CoA hydrolase in yeast cells involved in acetate utilization and subsequently affected the sporulation process.

Acetates↗

The dopamine transporter carboxyl-terminal tail. Truncation/substitution mutants selectively confer high affinity dopamine uptake while attenuating recognition of the ligand binding domain.

In order to delineate structural motifs regulating substrate affinity and recognition for the human dopamine transporter (DAT), we assessed [3H]dopamine uptake kinetics and [3H]CFT binding characteristics of COS-7 cells transiently expressing mutant DATs in which the COOH terminus was truncated or substituted. Complete truncation of the carboxyl tail from Ser582 allowed for the expression of biphasic [3H]dopamine uptake kinetics displaying both a low capacity (Vmax approximately 0.4 pmol/10(5) cells/min) high affinity (Km approximately 300 nM) component and one exhibiting low affinity (Km approximately 15 microM] and high capacity (Vmax approximately 5 pmol/10(5)cells/min) with a concomitant 40% decrease in overall apparent Vmax relative to wild type (WT) DAT. Truncation of the last 22 amino acids or substitution of the DAT-COOH tail with sequences encoding the intracellular carboxyl-terminal of either dopamine D1 or D5 receptors produced results that were identical to those with the fully truncated DAT, suggesting that the induction of biphasic dopamine uptake kinetics is likely conferred by removal of DAT-specific sequence motifs distal to Pro597. The attenuation of WT transport activity, either by lowering levels of DAT expression or by pretreatment of cells with phorbol 12-myristate 13-acetate (1 microM), did not affect the kinetics of [3H]dopamine transport. The estimated affinity of dopamine (Ki approximately 180 nM) for all truncated/substituted DAT mutants was 10-fold lower than that of WT DAT (approximately 2000 nM) and appears selective for the endogenous substrate, since the estimated inhibitory constants for numerous putative substrates or uptake inhibitors were virtually identical to those obtained for WT DATs. In marked contrast, DAT truncation/substitution mutants displayed significantly reduced high affinity [3H]CFT binding interactions with estimated Ki values for dopamine and numerous other substrates and inhibitors tested from 10-100-fold lower than that observed for WT DAT. Moreover, co-expression of truncated and/or substituted DATs with WT transporter failed to reconstitute functional or pharmacological activities associated with both transporters. Instead, complete restoration of uniphasic low affinity [3H]dopamine uptake kinetics (Km approximately 2000 nM) and high affinity substrate and inhibitor [3H]CFT binding interactions attributable to WT DATs were evident. These data clearly suggest the functional independence and differential regulation of the dopamine translocation process from the characteristics exhibited by its ligand binding domain. The lack of functional phenotypic expression of mutant DAT activities in cells co-expressing WT transporter is consistent with the contention that native DATs may exist as multisubunit complexes, the formation and maintenance of which is dependent upon sequences encoded within the carboxyl tail.

Animals↗

Molecular and functional characterization of a partial cDNA encoding a novel chicken brain melatonin receptor.

An approach based on homology probing was used to clone a partial cDNA encoding a novel melatonin (ML) receptor (MLR) from chicken (Gallus domesticus) brain. Based on available deduced amino-acid sequence, the chicken MLR (cMLR) displayed greater sequence homology to the frog (Xenopus) MLR than cloned human/mammalian receptors, with overall identities of 73% and 66%, respectively. In order to gain functional expression, a chimeric frog/chicken (flc)MLR was constructed in which the 5' end of the cMLR, including the N-terminus, TM1 and part of the first intracellular loop was substituted by fMLR sequence. [125I]Iodo-ML bound with high affinity (Kd of approximately 35 pM) to COS-7 cells transiently expressing the flcMLR in a saturable and guanine nucleotide-sensitive manner with the following rank order of potency: 2-iodo-ML > ML > 6-Cl-ML > S20750 > 6-OH-ML > S20642 > S20753 > N-acetyl-5HT >> 5-HT. Estimated Ki values for these compounds at the flcMLR correlated well to those obtained in native chicken brain membranes. In line with the observed structural similarity to the fMLR, the flcMLR exhibited affinities for ML, 6-Cl-ML and 6-OH-ML approximately 10-fold lower than mammalian receptors. Functionally, opposing interactions between ML and dopamine receptor signal transduction pathways were observed with ML potently inhibiting dopamine D1A-receptor-mediated cAMP accumulation in cells (HEK-293) transiently co-expressing these receptors. cMLR mRNAs were found expressed in chicken brain and kidney with trace levels observed in the lung. The availability of cloned vertebrate MLRs distinct at both the amino acid and pharmacological level from their mammalian counterparts may now allow for the identification of those amino-acid residues and structural motifs that regulate ML-binding specificity and affinity.

Adenylyl Cyclase Inhibitors↗

The dopamine D1D receptor. Cloning and characterization of three pharmacologically distinct D1-like receptors from Gallus domesticus.

Three genomic clones encoding dopamine D1-like receptors were isolated from the avian species Gallus domesticus. Two of these genes encode proteins of 451 and 488 amino acids, which, based on deduced amino acid sequence identity and homology of exhibited pharmacological profiles, appear to be species homologs of mammalian and vertebrate D1/D1A and D5/D1B receptors, respectively. The third genomic clone, termed D1D, encodes a protein of 445 amino acids displaying a deduced amino acid sequence identity within putative transmembrane domains of 75% to mammalian D1/D1A and 77% to D5/D1B receptors with overall sequence homologies of only 49% and 46%, respectively. Membranes from COS-7 cells transfected with D1D DNA bound [3H]SCH-23390 in a saturable manner with high affinity (approximately 300 pM) and with a pharmacological profile clearly indicative of a dopamine D1-like receptor. The D1D receptor exhibited affinities for 6,7-dihydroxy-2-aminotetralin and dopamine 10-fold higher than D1/D1A receptors, characteristic of the D5/D1B receptor subfamily. In contrast, the D1D receptor bound dopaminergic agents, such as SKF-38393, apomorphine, pergolide, and lisuride, with affinities 10-fold higher than other cloned mammalian or vertebrate D1A/D1B receptor subtypes, while both clozapine and haloperidol displayed considerably lower affinity for the D1D receptor. Based on the low overall amino acid sequence identity (54%) and unique pharmacological profile, the avian dopamine D1D receptor does not appear to be a species homolog of the recently cloned vertebrate D1C receptor (Sugamori, K.S., Demchyshyn, L. L., Chung, M., and Niznik, H. B. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10536-10540). As with all cloned mammalian and vertebrate D1-like receptors, the D1D receptor stimulates adenylate cyclase activity in the presence of dopamine or SKF-82526. Northern blot analysis reveals the selective expression of both avian D1D and D1A receptor mRNAs only in brain with the D1B receptor more widely distributed and localized in tissues such as brain, kidney, and spleen. The isolation of four distinct vertebrate dopamine D1 receptor subtypes suggests the existence of additional mammalian D1 like receptor genes that may account for the observed pharmacological and biochemical multiplicity of dopamine D1-like receptor mediated events.

Amino Acid Sequence↗

Different ARF domains are required for the activation of cholera toxin and phospholipase D.

ADP-ribosylation factors (ARFs), initially described as activators of cholera toxin ADP-ribosyltransferase activity, regulate intracellular vesicular membrane trafficking and stimulate a phospholipase D (PLD) isoform. ARF-like (ARL) proteins are structurally related to ARFs but do not activate cholera toxin and have relatively little effect on PLD. A new human ARL gene termed hARL1, which shares 57% amino acid identity with hARF1, was identified using a polymerase chain reaction-based cloning method. To determine whether different structural elements are responsible for the activation structural elements are responsible for the activation of the A subunit of cholera toxin and PLD, chimeric proteins were constructed by switching the amino-terminal 73 amino acids of ARF1 and ARL1. The recombinant rL73/F protein, in which the amino-terminal 73 amino acids of ARL1 replaced those of ARF1, activated the A subunit of cholera toxin, whereas the rF73/L protein, in which the NH2-terminal 73 amino acids of ARF1 replaced those of ARL1, was inactive. The two chimeric proteins had quite opposite effects on PLD activity. rF73/L activated PLD as effectively as rARF1, whereas rL73/F protein activated PLD only slightly. It appears that the amino-terminal region of ARF1 is not critical for its action as a GTP-dependent activator of cholera toxin, whereas it is necessary for activation of the putative effector enzyme, PLD.

ADP-Ribosylation Factor 1↗