Agouti-Related Protein Is Posttranslationally Cleaved by Proprotein Convertase 1 to Generate Agouti-Related Protein (AGRP)83–132: Interaction between AGRP83–132 and Melanocortin Receptors Cannot Be Influenced by Syndecan-3

Agouti-related protein (AGRP) plays a key role in energy homeostasis. The carboxyl-terminal domain of AGRP acts as an endogenous antagonist of the melanocortin-4 receptor (MC4-R). It has been suggested that the amino-terminal domain of AGRP binds to syndecan-3, thereby modulating the effects of carboxyl-terminal AGRP at the MC4-R. This model assumes that AGRP is secreted as a full-length peptide. In this study we found that AGRP is processed intracellularly after Arg79-Glu80-Pro81-Arg82. The processing site suggests cleavage by proprotein convertases (PCs). RNA interference and overexpression experiments showed that PC1/3 is primarily responsible for cleavage in vitro, although both PC2 and PC5/6A can also process AGRP. Dual in situ hybridization demonstrated that PC1/3 is expressed in AGRP neurons in the rat hypothalamus. Moreover, hypothalamic extracts from PC1-null mice contained 3.3-fold more unprocessed full-length AGRP, compared with wild-type mice, based on combined HPLC and RIA analysis, demonstrating that PC1/3 plays a role in AGRP cleavage in vivo. We also found that AGRP83–132 is more potent an antagonist than full-length AGRP, based on cAMP reporter assays, suggesting that posttranslational cleavage is required to potentiate the effect of AGRP at the MC4-R. Because AGRP is cleaved into distinct amino-terminal and carboxyl-terminal peptides, we tested whether amino-terminal peptides modulate food intake. However, intracerebroventricular injection of rat AGRP25–47 and AGRP50–80 had no effect on body weight, food intake, or core body temperature. Because AGRP is cleaved before secretion, syndecan-3 must influence food intake independently of the MC4-R.

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In vitro reconstitution of a heterotrimeric nucleoporin complex consisting of recombinant Nsp1p, Nup49p, and Nup57p.

Molecular Biology of the Cell ◽

10.1091/mbc.8.1.33 ◽

1997 ◽

Vol 8 (1) ◽

pp. 33-46 ◽

Cited By ~ 43

Author(s):

N L Schlaich ◽

M Häner ◽

A Lustig ◽

U Aebi ◽

E C Hurt

Keyword(s):

Nucleocytoplasmic Transport ◽

Full Length ◽

Yeast Cells ◽

Heptad Repeat ◽

Nuclear Pores ◽

Terminal Domain ◽

Carboxyl Terminal Domain ◽

Carboxyl Terminal

The yeast nucleoporins Nsp1p, Nup49p, and Nup57p form a complex at the nuclear pores which is involved in nucleocytoplasmic transport. To investigate the molecular basis underlying complex formation, recombinant full-length Nup49p and Nup57p and the carboxyl-terminal domain of Nsp1p, which lacks the FXFG repeat domain, were expressed in Escherichia coli. When the three purified proteins were mixed together, they spontaneously associated to form a 150-kDa complex of 1:1:1 stoichiometry. In this trimeric complex, Nup57p fulfills the role of an organizing center, to which Nup49p and Nsp1p individually bind. For this interaction to occur, only two heptad repeat regions of the Nsp1p carboxyl-terminal domain are required, each region being about 50 amino acids in length. Finally, the reconstituted complex has the capability to bind to full-length Nic96p but not to mutant forms which also do not interact in vivo. When added to permeabilized yeast cells, the complex associates with the nuclear envelope and the nuclear pores. We conclude that Nsp1p, Nup49p, and Nup57p can reconstitute a complex in vitro which is competent for further assembly with other components of nuclear pores.

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ADA3, a putative transcriptional adaptor, consists of two separable domains and interacts with ADA2 and GCN5 in a trimeric complex.

Molecular and Cellular Biology ◽

10.1128/mcb.15.3.1203 ◽

1995 ◽

Vol 15 (3) ◽

pp. 1203-1209 ◽

Cited By ~ 104

Author(s):

J Horiuchi ◽

N Silverman ◽

G A Marcus ◽

L Guarente

Keyword(s):

Activation Domains ◽

Amino Terminal ◽

Terminal Domain ◽

Trimeric Complex ◽

Nonoverlapping Domains ◽

Carboxyl Terminal Domain ◽

Carboxyl Terminal ◽

Amino Terminal Domain

Mutations in yeast ADA2, ADA3, and GCN5 weaken the activation potential of a subset of acidic activation domains. In this report, we show that their gene products form a heterotrimeric complex in vitro, with ADA2 as the linchpin holding ADA3 and GCN5 together. Further, activation by LexA-ADA3 fusions in vivo are regulated by the levels of ADA2. Combined with a prior observation that LexA-ADA2 fusions are regulated by the levels of ADA3 (N. Silverman, J. Agapite, and L. Guarente, Proc. Natl. Acad. Sci. USA 91:11665-11668, 1994), this finding suggests that these proteins also form a complex in cells. ADA3 can be separated into two nonoverlapping domains, an amino-terminal domain and a carboxyl-terminal domain, which do not separately complement the slow-growth phenotype or transcriptional defect of a delta ada3 strain but together supply full complementation. The carboxyl-terminal domain of ADA3 alone suffices for heterotrimeric complex formation in vitro and activation of LexA-ADA2 in vivo. We present a model depicting the ADA complex as a coactivator in which the ADA3 amino-terminal domain mediates an interaction between activation domains and the ADA complex.

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In vivo effect of sodium orthovanadate on pp60c-src kinase

Molecular and Cellular Biology ◽

10.1128/mcb.7.3.1139-1147.1987 ◽

1987 ◽

Vol 7 (3) ◽

pp. 1139-1147

Author(s):

J W Ryder ◽

J A Gordon

Keyword(s):

Tyrosine Kinase ◽

Chicken Embryo ◽

Sodium Orthovanadate ◽

Normal Cells ◽

Amino Terminal ◽

Carboxyl Terminal ◽

Embryo Fibroblasts ◽

Chicken Embryo Fibroblasts

We have compared the tyrosine kinase activity of pp60c-src isolated from intact chicken embryo fibroblasts treated with micromolar sodium orthovanadate for 4 h and from untreated cells. We found an approximate 50% reduction in both autophosphorylation of pp60c-src and phosphorylation of casein when examined in the immune complex kinase assay. The reduction of in vitro enzymatic activity correlated with a vanadate-induced increase in in vivo phosphorylation of pp60c-src at the major site of tyrosine phosphorylation in the carboxyl-terminal half of the molecule and at serine in the amino-terminal half of the molecule. Our observations in vivo and those of Courtneidge in vitro (EMBO J. 4:1471-1477, 1985) suggest that vanadate may enhance a cellular regulatory mechanism that inhibits the activity of pp60c-src in normal cells. A likely candidate for this mechanism is phosphorylation at a tyrosine residue distinct from tyrosine 416, probably tyrosine 527 in the carboxyl-terminal sequence of amino acids unique to pp60c-src. The regulatory role, if any, of serine phosphorylation in pp60c-src remains unclear. The 36-kilodalton phosphoprotein, a substrate of pp60v-src, showed a significant phosphorylation at tyrosine after treatment of normal chicken embryo fibroblasts with vanadate. Assuming that pp60c-src is inhibited intracellularly by vanadate, either another tyrosine kinase is stimulated by vanadate (e.g., a growth factor receptor) or the 36-kilodalton phosphoprotein in normal cells is no longer rapidly dephosphorylated by a tyrosine phosphatase in the presence of vanadate.

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The amino-terminal domain of pyrrolysyl-tRNA synthetase is dispensable in vitro but required for in vivo activity

FEBS Letters ◽

10.1016/j.febslet.2007.06.004 ◽

2007 ◽

Vol 581 (17) ◽

pp. 3197-3203 ◽

Cited By ~ 35

Author(s):

Stephanie Herring ◽

Alexandre Ambrogelly ◽

Sarath Gundllapalli ◽

Patrick O'Donoghue ◽

Carla R. Polycarpo ◽

...

Keyword(s):

Trna Synthetase ◽

Amino Terminal ◽

Terminal Domain ◽

Amino Terminal Domain

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Paraventricular Nucleus Administration of Calcitonin Gene-Related Peptide Inhibits Food Intake and Stimulates the Hypothalamo-Pituitary-Adrenal Axis

Endocrinology ◽

10.1210/en.2002-220902 ◽

2003 ◽

Vol 144 (4) ◽

pp. 1420-1425 ◽

Cited By ~ 42

Author(s):

Waljit S. Dhillo ◽

Caroline J. Small ◽

Preeti H. Jethwa ◽

Sabina H. Russell ◽

James V. Gardiner ◽

...

Keyword(s):

Food Intake ◽

Hpa Axis ◽

Male Rats ◽

Mrna Levels ◽

Related Protein ◽

Melanocyte Stimulating Hormone ◽

Calcitonin Gene ◽

Agouti Related Protein ◽

Prior Administration

Abstract Calcitonin gene-related protein (CGRP) inhibits food intake and stimulates the hypothalamo-pituitary-adrenal (HPA) axis after intracerebroventricular injection in rats. However, the hypothalamic site and mechanism of action are unknown. We investigated the effects of intraparaventricular nucleus administration (iPVN) of CGRP on food intake and the HPA axis in rats and the effect of CGRP on the release of hypothalamic neuropeptides in vitro. In addition, we investigated the effects of food deprivation on hypothalamic CGRP expression. CGRP dose-dependently reduced food intake in the first hour after iPVN injection in fasted male rats (saline, 5.1 ± 0.8 g; 0.3 nmol CGRP, 1.1 ± 0.5 g; P < 0.001 vs. saline). iPVN injection of CGRP8–37 (a CGRP1 receptor antagonist) alone had no effect on food intake. However, the reduction in food intake by iPVN CGRP was attenuated by prior administration of CGRP8–37 [CGRP8–37 (10 nmol)/CGRP (0.3 nmol), 3.0 ± 0.8 g; P < 0.05 vs. 0.3 nmol CGRP]. CGRP (100 nm) stimulated the release of α-melanocyte stimulating hormone, cocaine- and amphetamine-related transcript, corticotropin-releasing hormone, and arginine vasopressin from hypothalamic explants to 127 ± 19%, 148 ± 10%, 158 ± 17%, and 198 ± 21% of basal levels, respectively (P < 0.05 vs. basal), but did not alter the release of either neuropeptide Y or agouti-related protein. Hypothalamic CGRP mRNA levels in 24-h fasted rats were increased to 130 ± 8% of control levels [CGRP mRNA (arbitrary units), 4.75 ± 0.4; controls, 3.65 ± 0.34; P < 0.05]. Our data suggest that CGRP administered to the PVN inhibits food intake and stimulates the HPA axis.

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Identification of a cyclase-associated protein (CAP) homologue in Dictyostelium discoideum and characterization of its interaction with actin.

Molecular Biology of the Cell ◽

10.1091/mbc.7.2.261 ◽

1996 ◽

Vol 7 (2) ◽

pp. 261-272 ◽

Cited By ~ 50

Author(s):

U Gottwald ◽

R Brokamp ◽

I Karakesisoglou ◽

M Schleicher ◽

A A Noegel

Keyword(s):

Plasma Membrane ◽

Adenylyl Cyclase ◽

Dictyostelium Discoideum ◽

Direct Interaction ◽

Actin Binding ◽

Amino Terminal ◽

Terminal Domain ◽

Carboxyl Terminal Domain ◽

Carboxyl Terminal

In search for novel actin binding proteins in Dictyostelium discoideum we have isolated a cDNA clone coding for a protein of approximately 50 kDa that is highly homologous to the class of adenylyl cyclase-associated proteins (CAP). In Saccharomyces cerevisiae the amino-terminal part of CAP is involved in the regulation of the adenylyl cyclase whereas the loss of the carboxyl-terminal domain results in morphological and nutritional defects. To study the interaction of Dictyostelium CAP with actin, the complete protein and its amino-terminal and carboxyl-terminal domains were expressed in Escherichia coli and used in actin binding assays. CAP sequestered actin in a Ca2+ independent way. This activity was localized to the carboxyl-terminal domain. CAP and its carboxyl-terminal domain led to a fluorescence enhancement of pyrene-labeled G-actin up to 50% indicating a direct interaction, whereas the amino-terminal domain did not enhance. In polymerization as well as in viscometric assays the ability of the carboxyl-terminal domain to sequester actin and to prevent F-actin formation was approximately two times higher than that of intact CAP. The sequestering activity of full length CAP could be inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), whereas the activity of the carboxyl-terminal domain alone was not influenced, suggesting that the amino-terminal half of the protein is required for the PIP2 modulation of the CAP function. In profilin-minus cells the CAP concentration is increased by approximately 73%, indicating that CAP may compensate some profilin functions in vivo. In migrating D. discoideum cells CAP was enriched at anterior and posterior plasma membrane regions. Only a weak staining of the cytoplasm was observed. In chemotactically stimulated cells the protein was very prominent in leading fronts. The data suggest an involvement of D. discoideum CAP in microfilament reorganization near the plasma membrane in a PIP2-regulated manner.

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The Biochemical Activities of the Saccharomyces cerevisiae Pif1 Helicase Are Regulated by Its N-Terminal Domain

Genes ◽

10.3390/genes10060411 ◽

2019 ◽

Vol 10 (6) ◽

pp. 411 ◽

Cited By ~ 6

Author(s):

Nickens ◽

Sausen ◽

Bochman

Keyword(s):

Saccharomyces Cerevisiae ◽

Telomerase Activity ◽

Full Length ◽

Dna Unwinding ◽

Genome Maintenance ◽

Terminal Domain ◽

Pif1 Helicase ◽

Telomerase Regulation

: Pif1 family helicases represent a highly conserved class of enzymes involved in multiple aspects of genome maintenance. Many Pif1 helicases are multi-domain proteins, but the functions of their non-helicase domains are poorly understood. Here, we characterized how the N-terminal domain (NTD) of the Saccharomyces cerevisiae Pif1 helicase affects its functions both in vivo and in vitro. Removal of the Pif1 NTD alleviated the toxicity associated with Pif1 overexpression in yeast. Biochemically, the N-terminally truncated Pif1 (Pif1ΔN) retained in vitro DNA binding, DNA unwinding, and telomerase regulation activities, but these activities differed markedly from those displayed by full-length recombinant Pif1. However, Pif1ΔN was still able to synergize with the Hrq1 helicase to inhibit telomerase activity in vitro, similar to full-length Pif1. These data impact our understanding of Pif1 helicase evolution and the roles of these enzymes in the maintenance of genome integrity.

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A reassessment of copper(II) binding in the full-length prion protein

Biochemical Journal ◽

10.1042/bj20060458 ◽

2006 ◽

Vol 399 (3) ◽

pp. 435-444 ◽

Cited By ~ 57

Author(s):

Mark A. Wells ◽

Graham S. Jackson ◽

Samantha Jones ◽

Laszlo L. P. Hosszu ◽

C. Jeremy Craven ◽

...

Keyword(s):

Prion Protein ◽

Metal Ion ◽

Tandem Repeats ◽

Binding Mode ◽

Full Length ◽

Alternative Mode ◽

Histidine Residues ◽

Terminal Domain

It has been shown previously that the unfolded N-terminal domain of the prion protein can bind up to six Cu2+ ions in vitro. This domain contains four tandem repeats of the octapeptide sequence PHGGGWGQ, which, alongside the two histidine residues at positions 96 and 111, contribute to its Cu2+ binding properties. At the maximum metal-ion occupancy each Cu2+ is co-ordinated by a single imidazole and deprotonated backbone amide groups. However two recent studies of peptides representing the octapeptide repeat region of the protein have shown, that at low Cu2+ availability, an alternative mode of co-ordination occurs where the metal ion is bound by multiple histidine imidazole groups. Both modes of binding are readily populated at pH 7.4, while mild acidification to pH 5.5 selects in favour of the low occupancy, multiple imidazole binding mode. We have used NMR to resolve how Cu2+ binds to the full-length prion protein under mildly acidic conditions where multiple histidine co-ordination is dominant. We show that at pH 5.5 the protein binds two Cu2+ ions, and that all six histidine residues of the unfolded N-terminal domain and the N-terminal amine act as ligands. These two sites are of sufficient affinity to be maintained in the presence of millimolar concentrations of competing exogenous histidine. A previously unknown interaction between the N-terminal domain and a site on the C-terminal domain becomes apparent when the protein is loaded with Cu2+. Furthermore, the data reveal that sub-stoichiometric quantities of Cu2+ will cause self-association of the prion protein in vitro, suggesting that Cu2+ may play a role in controlling oligomerization in vivo.

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In vivo effect of sodium orthovanadate on pp60c-src kinase.

Molecular and Cellular Biology ◽

10.1128/mcb.7.3.1139 ◽

1987 ◽

Vol 7 (3) ◽

pp. 1139-1147 ◽

Cited By ~ 12

Author(s):

J W Ryder ◽

J A Gordon

Keyword(s):

Tyrosine Kinase ◽

Chicken Embryo ◽

Sodium Orthovanadate ◽

Normal Cells ◽

Amino Terminal ◽

Carboxyl Terminal ◽

Embryo Fibroblasts ◽

Chicken Embryo Fibroblasts

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Involvement of ZO-1 in Cadherin-based Cell Adhesion through Its Direct Binding to α Catenin and Actin Filaments

The Journal of Cell Biology ◽

10.1083/jcb.138.1.181 ◽

1997 ◽

Vol 138 (1) ◽

pp. 181-192 ◽

Cited By ~ 452

Author(s):

Masahiko Itoh ◽

Akira Nagafuchi ◽

Seiji Moroi ◽

Shoichiro Tsukita

Keyword(s):

Cell Adhesion ◽

Dissociation Constant ◽

Actin Filaments ◽

Full Length ◽

Amino Terminal ◽

Adhesion Sites ◽

Direct Binding ◽

Carboxyl Terminal ◽

E Cadherin

ZO-1, a 220-kD peripheral membrane protein consisting of an amino-terminal half discs large (dlg)-like domain and a carboxyl-terminal half domain, is concentrated at the cadherin-based cell adhesion sites in non-epithelial cells. We introduced cDNAs encoding the full-length ZO-1, its amino-terminal half (N-ZO-1), and carboxyl-terminal half (C-ZO-1) into mouse L fibroblasts expressing exogenous E-cadherin (EL cells). The full-length ZO-1 as well as N-ZO-1 were concentrated at cadherin-based cell–cell adhesion sites. In good agreement with these observations, N-ZO-1 was specifically coimmunoprecipitated from EL transfectants expressing N-ZO-1 (NZ-EL cells) with the E-cadherin/α, β catenin complex. In contrast, C-ZO-1 was localized along actin stress fibers. To examine the molecular basis of the behavior of these truncated ZO-1 molecules, N-ZO-1 and C-ZO-1 were produced in insect Sf9 cells by recombinant baculovirus infection, and their direct binding ability to the cadherin/catenin complex and the actin-based cytoskeleton, respectively, were examined in vitro. Recombinant N-ZO-1 bound directly to the glutathione-S-transferase fusion protein with α catenin, but not to that with β catenin or the cytoplasmic domain of E-cadherin. The dissociation constant between N-ZO-1 and α catenin was ∼0.5 nM. On the other hand, recombinant C-ZO-1 was specifically cosedimented with actin filaments in vitro with a dissociation constant of ∼10 nM. Finally, we compared the cadherin-based cell adhesion activity of NZ-EL cells with that of parent EL cells. Cell aggregation assay revealed no significant differences among these cells, but the cadherin-dependent intercellular motility, i.e., the cell movement in a confluent monolayer, was significantly suppressed in NZ-EL cells. We conclude that in nonepithelial cells, ZO-1 works as a cross-linker between cadherin/catenin complex and the actin-based cytoskeleton through direct interaction with α catenin and actin filaments at its amino- and carboxyl-terminal halves, respectively, and that ZO-1 is a functional component in the cadherin-based cell adhesion system.

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