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1.
Biochemistry ; 40(39): 11734-41, 2001 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-11570874

RESUMO

Prostate specific antigen (PSA, also known as human kallikrein 3) is an important diagnostic indicator of prostatic disease. PSA exhibits low protease activity (>10(4)-fold less than chymotrypsin) under the usual in vitro assay conditions. In addition, PSA does not react readily with prototypical serine protease inactivators. We expressed human PSA (rh-PSA) in Escherichia coli and have demonstrated that rh-PSA has properties similar to those of native PSA isolated from human seminal fluid. Both PSA and rh-PSA are >10(3)-fold more active in the presence of 1.3 M Na(2)SO(4). This activation is anion-dependent, following the Hofmeister series when normality is considered: SO(4)(2)(-) approximately citrate > Ac(-) > Cl(-) > Br(-) > I(-). The nature of the cation has little effect on salt activation. The rate of inactivation of rh-PSA by DFP is 30-fold faster in the presence of 0.9 M Na(2)SO(4), and the rate of inactivation by Suc-Ala-Ala-Pro-Phe-CK is >20-fold faster under these conditions. Azapeptides containing Phe or Tyr at position P(1) also inactivate rh-PSA in the presence of high salt concentrations. These compounds represent the first described inhibitors designed to utilize the substrate binding subsites of PSA. CD spectroscopy demonstrates that the conformation of rh-PSA changes in the presence of high salt concentrations. Analytical ultracentifugation and dynamic light scattering indicate that PSA remains monomeric under high-salt conditions. Interestingly, human prostatic fluid contains as much as 150 micro mol citrate/g wet weight, which suggests that salt concentrations may regulate PSA activity in vivo.


Assuntos
Antígeno Prostático Específico/metabolismo , Sequência de Bases , Primers do DNA , Escherichia coli/genética , Humanos , Espectrometria de Massas , Ressonância Magnética Nuclear Biomolecular , Antígeno Prostático Específico/química , Antígeno Prostático Específico/genética , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
2.
Biochem Biophys Res Commun ; 285(3): 742-50, 2001 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-11453656

RESUMO

Despite attempts in several laboratories, it has been difficult to prepare antiserum to the thyrotropin-releasing hormone receptor (TRHR). We have prepared a polyclonal anti-rat TRHR antiserum by immunization of rabbits with a synthetic peptide corresponding to the C-terminus of the TRHR. The specificity of the antiserum was assessed by enzyme-linked immunosorbent assay. The affinity-purified antibody recognized a major broad band at 50-60 kDa and a minor broad band at 100-120 kDa in Western blot analysis of membrane proteins from TRHR-transfected, but not control, HEK293t cells. Binding to both bands was abolished by preincubation with the immunizing peptide but not control peptide. The approach was repeated with rat pituitary F4C1 cells, which lack endogenous TRHRs; membranes from F4C1 cells transfected with TRHR cDNA, but not control cells, showed specific binding by Western blot. Using laser confocal microscopy, the TRHR was visualized on the plasma membrane of transfected, but not control, F4C1 cells. Similar confocal findings were observed in TRHR-transfected HEK293t cells. Within 5 min after TRH addition, the TRHR signal translocated from the plasma membrane to the cytoplasm of F4C1 cells transfected with TRHR cDNA. Ten minutes after TRH addition, the TRHR signal formed aggregates in the cytoplasm. Thirty minutes after TRH treatment, both cytoplasmic and plasma membrane localizations were observed, suggesting recycling of some TRHRs back to the plasma membrane. These observations are consistent with our previous findings using an epitope-tagged TRHR. In conclusion, we have prepared an antiserum that recognizes the native TRHR by Western blot analysis and confocal microscopy.


Assuntos
Especificidade de Anticorpos/imunologia , Soros Imunes/química , Soros Imunes/isolamento & purificação , Receptores do Hormônio Liberador da Tireotropina/imunologia , Animais , Ligação Competitiva/imunologia , Western Blotting , Linhagem Celular , Membrana Celular/metabolismo , DNA Complementar/genética , DNA Complementar/metabolismo , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Humanos , Soros Imunes/metabolismo , Imuno-Histoquímica , Técnicas de Imunoadsorção , Microscopia Confocal , Hipófise/citologia , Hipófise/metabolismo , Coelhos , Ratos , Receptores do Hormônio Liberador da Tireotropina/genética , Receptores do Hormônio Liberador da Tireotropina/metabolismo , Transfecção
3.
J Biol Chem ; 275(35): 27238-44, 2000 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-10837469

RESUMO

The N-terminal fragment 1-34 of parathyroid hormone (PTH), administered intermittently, results in increased bone formation in patients with osteoporosis. PTH and a related molecule, parathyroid hormone-related peptide (PTHrP), act on cells via a common PTH/PTHrP receptor. To define more precisely the ligand-receptor interactions, we have crystallized human PTH (hPTH)-(1-34) and determined the structure to 0.9-A resolution. hPTH-(1-34) crystallizes as a slightly bent, long helical dimer. Analysis reveals that the extended helical conformation of hPTH-(1-34) is the likely bioactive conformation. We have developed molecular models for the interaction of hPTH-(1-34) and hPTHrP-(1-34) with the PTH/PTHrP receptor. A receptor binding pocket for the N terminus of hPTH-(1-34) and a hydrophobic interface with the receptor for the C terminus of hPTH-(1-34) are proposed.


Assuntos
Hormônio Paratireóideo/química , Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Hormônio Paratireóideo/metabolismo , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Receptores de Hormônios Paratireóideos/metabolismo , Homologia de Sequência de Aminoácidos
4.
J Bone Miner Res ; 15(3): 550-6, 2000 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10750570

RESUMO

Previous findings have shown that osteoblasts respond to parathyroid hormone (PTH) with an increase in extracellular acidification rate (ECAR) in addition to the known effect of PTH to increase local acidification by osteoclasts. We, therefore, investigated use of the Cytosensor to measure the ECAR response of whole intact bone to PTH employing microphysiometry. The Cytosensor measures a generic metabolic increase of cells to various agents. Using neonatal mouse calvaria, we found that the area surrounding the sagittal suture was particularly responsive to PTH. In this bone, the increase in ECAR was slower to develop (6 minutes) and more persistent than in cultured human osteoblast-like SaOS-2 cells and was preceded by a brief decrease in ECAR. Salmon calcitonin also produced an increase in ECAR in this tissue but with a different pattern than that elicited by PTH. Because PTH stimulates osteoclastic bone resorption in mouse calvaria via a cyclic adenosine monophosphate (cAMP)-mediated mechanism, we showed that the adenylyl cyclase activator forskolin also stimulated ECAR in this tissue. When the protein kinase A (PKA) pathway was activated by maintaining a high intracellular concentration of cAMP using N6-2'-0-dibutyryladenosine-cAMP (db-cAMP), there was a reduction of PTH-induced acidification, while isobutylmethylxanthine pretreatment potentiated the PTH-induced acidification, consistent with a PKA-mediated pathway. Thapsigargin and the protein kinase C (PKC) activator phorbol myristate acetate had no effect on the PTH-induced increase in ECAR in calvaria, indicating that PKC does not play a major role in the ECAR response in intact bone. These results indicate the utility of using microphysiometry to study ECAR responses in intact tissue and should enable elucidation of the relative importance of extracellular acidification by osteoblasts and osteoclasts to the anabolic and catabolic activities of PTH, respectively.


Assuntos
Osso e Ossos/efeitos dos fármacos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Hormônio Paratireóideo/farmacologia , 1-Metil-3-Isobutilxantina/farmacologia , Adenilil Ciclases/fisiologia , Animais , Animais Recém-Nascidos , Neoplasias Ósseas/patologia , Osso e Ossos/química , Bucladesina/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Anidrases Carbônicas/fisiologia , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Ativação Enzimática/efeitos dos fármacos , Espaço Extracelular/química , Espaço Extracelular/efeitos dos fármacos , Humanos , Camundongos , Técnicas de Cultura de Órgãos , Especificidade de Órgãos , Osteoblastos/metabolismo , Osteossarcoma/patologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/fisiologia , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Crânio/química , Crânio/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Tapsigargina/farmacologia , Células Tumorais Cultivadas/química , Células Tumorais Cultivadas/efeitos dos fármacos
5.
Cell Signal ; 12(4): 255-63, 2000 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10781933

RESUMO

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the vasoactive intestinal peptide/secretin family. Using microphysiometry, we have found that PACAP acutely (1 min) increased the extracellular acidification rate (ECAR) in GH4C1 cells approximately 40% above basal in a concentration-dependent manner. ECAR, maximally induced by PACAP, can be increased further by thyrotropin-releasing hormone (TRH), indicating that the signalling pathways for these two neuropeptides are not identical. In studies on the mechanism of PACAP-enhanced ECAR, we found that maximum stimulation of the cAMP/PKA pathway by treatment with FSK, or the PKC pathway with PMA, did not inhibit the ECAR response to PACAP. The PKC inhibitor calphostin C and the MAP kinase inhibitor PD98059 had no effect on the ECAR response to PACAP. Furthermore, PACAP induced little or no change in cytosolic Ca(2+) ([Ca(2+)](i)), while TRH induced a large increase in [Ca(2+)](i). However, the tyrosine kinase inhibitor genistein completely blocked PACAP-induced ECAR, suggesting involvement of tyrosine kinase(s). We conclude that PACAP causes an increase in ECAR in GH4C1 rat pituitary cells, which is not dependent on the PKA, PKC, MAP kinase or Ca(2+) signalling pathways, but does require tyrosine kinase activity.


Assuntos
Neuropeptídeos/farmacologia , Hipófise/fisiologia , Transdução de Sinais/fisiologia , Animais , Cálcio/metabolismo , Linhagem Celular , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Espaço Extracelular/fisiologia , Flavonoides/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Naftalenos/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Hipófise/citologia , Proteína Quinase C/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Hormônio Liberador de Tireotropina/farmacologia
6.
Biochem Biophys Res Commun ; 266(2): 448-53, 1999 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-10600523

RESUMO

To examine the importance of the N- or C-termini of PTH(1-34) the effects of truncated fragments of PTH on human receptors in osteoblast-like SaOS-2 cells and rat receptors in rats ROS 17/2 cells were examined. Fura-2-loaded cells were used to monitor cytosolic free Ca(2+) concentration ([Ca2+]i), and the Cytosensor microphysiometer was used to monitor extracellular acidification rate (ECAR). C-terminally truncated fragments (1-31) and (1-28) of hPTH(1-34)NH(2) stimulated an increase in [Ca(2+)](i) and ECAR in both cell lines. hPTH(3-34)NH(2) and other N-terminally truncated fragments did not stimulate [Ca(2+)](i) or ECAR in either cell type. The signal transduction pathway of PTH-induced ECAR in ROS 17/2 cells was investigated to compare with previous results in SaOS-2 cells. Potentiation by IBMX, attenuation by 8Br-cAMP and lack of effect of the PKC inhibitor chelerythrine chloride support a cAMP/PKA-mediated signal transduction pathway in ROS 17/2, while the protein kinase C pathway was predominant in SaOS-2 cells. We conclude that the intact N-terminus of PTH is essential in PTH signaling mediated via either the cAMP/PKA or inositol lipid/Ca(2+)/PKC pathways in osteoblast-like cells.


Assuntos
Cálcio/metabolismo , Hormônio Paratireóideo/farmacologia , 1-Metil-3-Isobutilxantina/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Alcaloides , Animais , Benzofenantridinas , Linhagem Celular , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fura-2 , Humanos , Concentração de Íons de Hidrogênio , Cinética , Fragmentos de Peptídeos/farmacologia , Fenantridinas/farmacologia , Proteína Quinase C/metabolismo , Ratos , Transdução de Sinais , Espectrometria de Fluorescência , Acetato de Tetradecanoilforbol/farmacologia
7.
Cell Signal ; 11(7): 499-505, 1999 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10405760

RESUMO

Somatostatin receptors (SSTRs) are known to mediate diverse cellular responses. Most target cell express more than one SSTR isoform, making it difficult to define the signalling pathway used by individual receptor subtypes. Thus, we have expressed SSTR1 or SSTR2 in rat pituitary F4C1 cells which lack endogenous SSTRs. Using a silicon-based biosensor system, the Cytosensor microphysiometer, which measures the extracellular acidification rate (ECAR) in real time, we have studied the responses to SS mediated by either SSTR1 or SSTR2. In control F4C1 cells, SS had no effect on the basal ECAR. In transfected cells expressing only SSTR1, SS caused a unique decrease in ECAR in a concentration-dependent manner. Receptor-mediated decreases in ECAR have not been reported previously. In F4C1 cells expressing only SSTR2, SS induced a bidirectional ECAR response, a rapid increase followed by a decrease below basal. Two SS analogues, MK678 and CH275, induced characteristic ECAR responses with the expected receptor selectivities for SSTR1 or SSTR2. Pretreatment of F4C1 cells with pertussis toxin abolished the decreases in ECAR mediated by both SSTR1 and SSTR2, but only partially reduced the increase in ECAR mediated by SSTR2. The decrease in ECAR did not depend on a decrease in intracellular cAMP. The ECAR responses to SS were modestly attenuated by methylisobutylamiloride (MIA), an inhibitor of the ubiquitous Na(+)-H+ exchanger NHE1. Removal of extracellular Na+ greatly inhibited the ECAR responses to SS, demonstrating a role for both amiloride-sensitive and -insensitive Na(+)-dependent acid transport mechanisms in SS-induced extracellular acidification. In conclusion, we have identified and characterized different signalling pathways for SSTR1 and SSTR2 in pituitary cells as measured by microphysiometry.


Assuntos
Receptores de Somatostatina/metabolismo , Transdução de Sinais , Animais , AMP Cíclico/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Antagonistas de Hormônios/farmacologia , Peptídeos Cíclicos/farmacologia , Toxina Pertussis , Ratos , Trocadores de Sódio-Hidrogênio/fisiologia , Somatostatina/análogos & derivados , Somatostatina/farmacologia , Células Tumorais Cultivadas , Fatores de Virulência de Bordetella/farmacologia
8.
Biochem Biophys Res Commun ; 258(3): 689-94, 1999 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-10329447

RESUMO

The availability of subtype-specific agonists and antagonists for somatostatin (SS) receptors (SSTRs) will be important for elucidation of the function of each receptor isoform in vivo. A SS analog, des-AA1,2,5-[D-Trp8, IAmp9]SS (CH275), has been shown previously to bind preferentially to SSTR1. In this report, we identify structural determinants in the ligand and receptor responsible for the selective binding of CH275 to SSTR1 by modifying both the ligand and the receptor. We propose that IAmp9 in CH275, like Lys9 in SS, interacts with Asp137 in the middle of the third transmembrane domain of SSTR1 to form an ion pair, while other residues unique to SSTR1 conbribute to binding selectivity of CH275 for SSTR1. Replacement of Asp137 with Asn resulted in loss of binding of radiolabeled SS and decreased potencies of both SS and CH275 to induce a change in the extracellular acidification rate measured by microphysiometry. The structural determinants for specific binding to SSTR1 were mapped in chimeric SSTR1/SSTR2 receptors. One chimera, 2beta, with the N-terminus to second transmembrane domain (TM2) from SSTR2 and the remainder of the receptor from SSTR1, had low affinity for CH275. Furthermore, when a single residue, Leu107, in TM2 of SSTR1 was replaced with Phe, the corresponding residue in SSTR2, a 20-fold decrease in affinity for CH275 with no significant change in affinity for SS was observed. A reciprocal change from Phe to Leu in the chimeric receptor 2beta resulted in a 10-fold increase in affinity for CH275. Thus, Leu107 is an important determinant for CH275 binding to SSTR1. To identify the moiety in CH275 which could interact with Leu107, a new analog des-AA1,2,5-[D-Trp8, Amp9]SS was prepared. This analog bound to both SSTR1 and SSTR2 with similar affinities; thus, subtype selectivity was lost. Collectively, these data support a binding model for CH275 in which the positively charged IAmp interacts with the negatively charged Asp137 in TM3 of SSTR1 and the isopropyl group of IAmp forms a hydrophobic interaction with Leu107 in TM2.


Assuntos
Receptores de Somatostatina/metabolismo , Somatostatina/análogos & derivados , Animais , Ácido Aspártico/metabolismo , Linhagem Celular , Ligação Proteica , Conformação Proteica , Ratos , Somatostatina/química , Somatostatina/metabolismo
9.
J Bone Miner Res ; 14(2): 163-72, 1999 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-9933468

RESUMO

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are believed to exert their biological actions through binding and activation of a common cell surface receptor. Recently, an analog of PTHrP (RS-66271), was described that demonstrated reduced binding affinity for the PTH/PTHrP receptor compared with bovine PTH(1-34) but retained equal biological activity. The present study investigated the receptor binding affinities of synthetic RS-66271 and recombinant human PTH(1-34) (LY333334) and compared their in vitro and in vivo pharmacological effects. RS-66271 had one hundredth the activity of PTH(1-34) in competing for the binding of [125I] [Nle8,18, Tyr34]human PTH(1-34) to the human PTH/PTHrP receptor stably expressed in a human kidney cell line. Despite this reduced binding affinity, RS-66271 had equivalent activity in increasing both cAMP production in osteoblast-like cells and bone resorption in neonatal mouse calvariae. However, RS-66271 was 7. 6-fold less active in stimulating inositol phosphate production. For in vivo studies, young, male Fisher rats received a daily subcutaneous dose of either 10 or 40 microg/kg of peptide for 1, 2, or 4 weeks. Volumetric bone mineral density and total bone mineral content of the proximal tibia were determined by peripheral quantitative computerized tomography. Trabecular and cortical bone of the distal femur were analyzed for calcium and dry weight. Lumbar vertebrae (L4-L6) were analyzed by histomorphometry. Trabecular and cortical bone mass showed a dose- and time-dependent increase in the treated animals compared with the controls. These increases were evident as early as 1 week after initiation of dosing. There were no consistent significant differences in the comparative effects of PTH(1-34) and RS-66271 on the measured bone parameters. In conclusion, despite the reduced binding affinity of RS-66271 for the PTH/PTHrP receptor compared with human PTH(1-34), both peptides displayed similar in vitro and in vivo pharmacological effects.


Assuntos
Densidade Óssea/efeitos dos fármacos , Teriparatida/análogos & derivados , Teriparatida/farmacologia , Animais , Ligação Competitiva , Reabsorção Óssea/induzido quimicamente , Cálcio/metabolismo , Bovinos , Linhagem Celular , Humanos , Masculino , Camundongos , Técnicas de Cultura de Órgãos , Ratos , Ratos Endogâmicos F344 , Receptor Tipo 1 de Hormônio Paratireóideo , Receptores de Hormônios Paratireóideos/metabolismo , Transdução de Sinais , Teriparatida/metabolismo
10.
Oncogene ; 18(5): 1251-6, 1999 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-10022132

RESUMO

We have analysed the expression of three calcium-independent isoforms of protein kinase C (PKC), PKCdelta, PKCepsilon and PKCzeta, in an in vitro model of colon carcinogenesis consisting of the nontumorigenic rat colonic epithelial cell line D/WT, and a derivative src-transformed line D/src. While PKCzeta and PKCepsilon showed similar protein levels, PKCdelta was markedly decreased in D/src cells when compared to the D/WT line. To assess whether down-regulation of PKCdelta was causally involved in the neoplastic phenotype in D/src cells, we prepared a kinase-defective mutant of PKCdelta. Stable transfection of this sequence caused morphological and growth changes characteristic of partial transformation in D/WT cells. Moreover, to test whether PKCdelta was involved in growth control and transformation in this model, we overexpressed PKCdelta in D/src cells. Transfected cells underwent marked growth and morphological modifications toward the D/WT phenotype. In a late stage in culture, transfected cells ceased to proliferate, rounded up and degenerated into multinucleated, giant-like cells. We conclude that PKCdelta can reverse the transformed phenotype and act as a suppressor of cell growth in D/src cells. Moreover, our data show that downregulation of this isoenzyme of PKC may cooperate in the neoplastic transformation induced by the src oncogene in D/WT cells.


Assuntos
Transformação Celular Neoplásica , Neoplasias do Colo/enzimologia , Genes src , Inibidores do Crescimento/biossíntese , Isoenzimas/biossíntese , Proteína Quinase C/biossíntese , Animais , Cálcio/metabolismo , Neoplasias do Colo/genética , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Inibidores do Crescimento/genética , Mucosa Intestinal/enzimologia , Mucosa Intestinal/patologia , Isoenzimas/genética , Proteína Quinase C/genética , Proteína Quinase C-delta , Ratos , Proteínas Recombinantes/biossíntese
11.
Cell Signal ; 10(8): 553-9, 1998 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9794253

RESUMO

It has been proposed that, after agonist binding, the thyrotropin-releasing hormone receptor (TRHR) becomes internalised associated with Gq, as part of a TRH-TRHR-Gq ternary complex [13]. We tested this hypothesis directly by examining the intracellular distribution of the TRHR and Gq/11 after agonist binding. The localisation of the TRH-TRHR complex and Gq/11alpha was studied by the biochemical isolation of clathrin-coated vesicles (CCVs). The internalised TRH-TRHR complex was localised in CCVs. The CCVs, which had internalised [3H]MeTRH, contained 4-fold higher levels of radiolabelled ligand than did CCVs from cells incubated with [3H]MeTRH at 4 degrees C. Like the receptor-ligand (RL) complex, G11alpha also translocated to these endocytic vesicles. For example, CCVs from cells with internalised TRH-TRHR complexes contained G11alpha, whereas CCVs from cells without internalised RL complexes lacked G11alpha. We conclude that, after agonist-induced TRHR-G11alpha coupling, both the TRH-TRHR complex and G11alpha are internalised in CCVs.


Assuntos
Clatrina/metabolismo , Vesículas Revestidas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Receptores do Hormônio Liberador da Tireotropina/metabolismo , Hormônio Liberador de Tireotropina/metabolismo , Animais , Transporte Biológico Ativo , Linhagem Celular , Cinética , Substâncias Macromoleculares , Ratos , Transdução de Sinais
12.
Biotechniques ; 25(2): 256-9, 262-3, 1998 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9714886

RESUMO

We have developed a simple and flexible program to analyze regional hydrophobicity of a protein from its amino acid (aa) sequence. This program runs as a Microsoft Excel document into which aa sequence can be copied from any Windows-compatible or Macintosh word processor. The program returns the hydrophobicity index of each aa residue and other analyses that can be used to predict transmembrane domains, amphiphilic alpha helices and putative antigenic epitopes in a protein using established algorithms. The program can also be easily modified to test user-defined algorithms and to accommodate non-conventional aa residues or ambiguities in the aa sequence. Simple modification of the program allows direct use of nucleic acid sequence information for various analyses. Graphic visualization of the results is readily achieved using the graphics function of Microsoft Excel. Alternatively, the data can be imported into other graphics software for preparation of publication-quality figures. By running as a document in Microsoft Excel, which can be found in virtually all personal computers, this program provides easy access even to the computer novice.


Assuntos
Proteínas/química , Software , Sequência de Aminoácidos , Animais , Sequência de Bases , Computação Matemática , Dados de Sequência Molecular , Ratos , Receptores do Hormônio Liberador da Tireotropina/química
13.
Oncogene ; 16(25): 3345-8, 1998 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-9681835

RESUMO

We have shown previously that overexpression of the epsilon isoform of protein kinase C (PKCepsilon) in rat colonic epithelial cells causes malignant transformation, possibly by interacting with the ras signal transduction pathway (Oncogene 12: 847, 1996). We have now performed experiments to examine certain early steps in the ras signaling pathway. A marked increase of Raf-1 phosphorylation was detected in tumorigenic ras-transformed D/ras as well as in D/epsilon cells (overexpressing PKCepsilon), compared to the nontumorigenic D/WT parental line. Moreover, in the PKCepsilon-transformed D/epsilon cell line, stable transfection with a dominant-negative raf-1 (DNraf) sequence caused complete regression of the neoplastic phenotype. These results suggested that PKCepsilon-induced transformation was associated with increased Raf-1 activation, and that DNraf could block the oncogenic effect of PKCepsilon. Furthermore, transfection of D/WT cells with dominant-negative ras induced arrest of cell growth, and subsequent transfection with PKCepsilon cDNA enhanced cell proliferation and induced neoplastic transformation. These results suggest that ras acts upstream of PKCepsilon, and that overexpression of PKCepsilon circumvents the block in cell proliferation caused by dominant-negative ras. We conclude that PKCepsilon exerts its oncogenic activity in rat colonic cells by affecting the ras signaling cascade at the level of Raf-1 activation.


Assuntos
Colo/citologia , Células Epiteliais/metabolismo , Isoenzimas/fisiologia , Proteína Quinase C/fisiologia , Proteínas ras/fisiologia , Animais , Divisão Celular/genética , Divisão Celular/fisiologia , Linhagem Celular , Linhagem Celular Transformada , Transformação Celular Neoplásica/genética , Colo/metabolismo , Células Epiteliais/citologia , Genes ras/genética , Genes ras/fisiologia , Isoenzimas/genética , Mutação/genética , Mutação/fisiologia , Fosforilação , Proteína Quinase C/genética , Proteína Quinase C-épsilon , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteínas ras/genética
14.
Am J Physiol ; 274(2): E297-303, 1998 02.
Artigo em Inglês | MEDLINE | ID: mdl-9486161

RESUMO

We prepared a polyclonal antiserum [Ab-(88-97)] against residues 8-97 of the NH2-terminal tail of the human (h) parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor. Ab-(88-97) bound specifically to the receptor, as assessed by fluorescence-activated cell sorter analysis of HEK C21 cells, which stably express approximately 400,000 hPTH/PTHrP receptors per cell. Unlike PTH, Ab-(88-97) binding did not elicit either adenosine 3',5'-cyclic monophosphate or intracellular calcium concentration signaling responses in these cells. Incubation of C21 cells for 90 min at 4 degrees C with hPTH-(1-34) plus antiserum reduced the Ab-(88-97) binding to the cells by up to 40-50% of control values in a PTH concentration-dependent fashion with a half-maximal effective concentration of approximately 5 nM. The decrease in Ab-(88-97) binding caused by hPTH-(1-34) was completely reversed by coincubation with hPTHrP-(7-34). We conclude that residues 88-97 of the hPTH/PTHrPR are involved, either directly or indirectly, in agonist but not antagonist binding to the receptor.


Assuntos
Receptores de Hormônios Paratireóideos/metabolismo , Animais , Linhagem Celular , Mapeamento de Epitopos , Cabras , Humanos , Soros Imunes , Hormônio Paratireóideo/imunologia , Hormônio Paratireóideo/metabolismo , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Receptor Tipo 1 de Hormônio Paratireóideo , Receptores de Hormônios Paratireóideos/imunologia , Teriparatida/análogos & derivados , Teriparatida/imunologia , Teriparatida/metabolismo
15.
J Exp Med ; 187(5): 775-85, 1998 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-9480987

RESUMO

Microphthalmia (Mi) is a basic helix-loop-helix-leucine zipper (b-HLH-ZIP) transcription factor implicated in pigmentation, mast cells, and bone development. Two dominant-negative mi alleles (mi/mi and Mior/Mior) in mice cause osteopetrosis. In contrast, osteopetrosis has not been observed in a number of recessive mi alleles, suggesting the existence of Mi protein partners important in osteoclast function. An osteopetrotic rat of unknown genetic defect (mib) has been described whose skeletal sclerosis improves dramatically with age and that is associated with pigmentation defects reminiscent of mouse mi alleles. Here we report that this rat strain harbors a large genomic deletion encompassing the 3' half of mi including most of the b-HLH-ZIP region. Osteoclasts from these animals lack Mi protein in contrast to wild-type rat, mouse, and human osteoclasts. Mi is not detectable in primary osteoblasts. In addition TFE3, a b-HLH-ZIP transcription factor related to Mi, was found to be expressed in osteoclasts, but not osteoblasts, and to coimmunoprecipitate with Mi. These results demonstrate the existence of members of a family of biochemically related transcription factors that may cooperate to play a central role in osteoclast function and possibly in age-related osteoclast homeostasis.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Microftalmia/genética , Osteoclastos/fisiologia , Osteopetrose/etiologia , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Expressão Gênica , Sequências Hélice-Alça-Hélice , Humanos , Zíper de Leucina , Camundongos , Fator de Transcrição Associado à Microftalmia , Dados de Sequência Molecular , Osteoblastos/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Mutantes , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico
16.
Cell Signal ; 9(6): 469-74, 1997 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9376230

RESUMO

To investigate whether G protein-coupled receptor kinases (GRKs) are involved in the regulation of the PTH/PTHrPR, we have established mutant SaOS-2 cells which stably overexpress (> 10-20-fold) a dominant negative form of the beta-adrenergic receptor kinase-1 (beta ARK-1). Acute (< or = 2 h) incubation with hPTH (1-34) induced significantly less (by up to 50%) downregulation of the PTH/PTHrPR in beta ARK-1 mutant SaOS-2 cells than observed in wild-type cells. Pretreatment of wild-type cells with PTH for 2 h induced homologous cAMP desensitisation to a second challenge with PTH, while the effect was blunted by up to 60% in beta ARK-1 mutant cells. We conclude that activation of beta ARK-1 (or a closely related GRK) is a critical component of the acute phase (< or = 2 h) of PTH-induced receptor downregulation and homologous cAMP desensitisation of the PTH/PTHrPR.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Oncogênicas , Osteoblastos/enzimologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Hormônios Paratireóideos/fisiologia , Transdução de Sinais/fisiologia , Linhagem Celular , Colforsina/farmacologia , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/genética , Ativação Enzimática , Humanos , Mutação/fisiologia , Osteoblastos/citologia , Proteínas Proto-Oncogênicas , Receptor Tipo 1 de Hormônio Paratireóideo , Sensibilidade e Especificidade , Transdução de Sinais/efeitos dos fármacos , Transfecção , Quinases de Receptores Adrenérgicos beta , Receptor Tirosina Quinase Axl
17.
J Biol Chem ; 272(42): 26346-53, 1997 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-9334207

RESUMO

The major physiological function of parathyroid hormone (PTH) is the maintenance of Ca2+/Pi homeostasis via the parathyroid hormone/parathyroid hormone-related protein receptor (PTHR) in kidney and bone. An important consequence of PTHR activation in bone is enhanced local acidification of the extracellular space. Agonist activation of some seven transmembrane-domain receptors increases the extracellular acidification rate (ECAR). We utilized microphysiometry to investigate PTH-stimulated, receptor-mediated increases in ECAR in human osteoblast-like SaOS-2 cells. PTH-(1-34) elicited a large, acute, dose-dependent increase in ECAR with an EC50 of about 2 nM. The PTH-induced increase in ECAR was specific to cells expressing the PTHR and was inhibited by PTHR antagonists. Rapid, partial, homologous desensitization of the PTH-induced increase in ECAR was observed. Incubation of SaOS-2 cells with 8-bromo-cyclic AMP neither mimicked nor abrogated the PTH effect, and PTH stimulated an acute increase in ECAR in cAMP-resistant SaOS-2 Ca#4A cells. Stimulation of ECAR by PTH was independent of transient increases in cytosolic free calcium. Both inhibition and down-regulation of PKC reduced the PTH-induced increase in ECAR. Inhibition of Na+/H+ exchange did not affect the PTH-induced ECAR response. We conclude that PTH caused a receptor-mediated, concentration-dependent, increase in ECAR, which was not dependent on the cAMP/PKA signaling pathway or the Na+/H+ exchanger but involved the action of PKC. Thus, acid production in bone, a physiologically important action of PTH, is not confined to osteoclasts as previously considered but is also mediated by osteoblasts.


Assuntos
Osteoblastos/efeitos dos fármacos , Hormônio Paratireóideo/farmacologia , Receptores de Hormônios Paratireóideos/metabolismo , Linhagem Celular , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Espaço Extracelular/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Osteoblastos/metabolismo , Peptídeos/metabolismo , Transdução de Sinais , Trocadores de Sódio-Hidrogênio/metabolismo
18.
J Biol Chem ; 272(30): 18666-72, 1997 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-9228036

RESUMO

To elucidate the signaling events mediated by specific somatostatin receptor (SSTR) subtypes, we expressed SSTR1 and SSTR2 individually in rat pituitary GH12C1 and F4C1 cells, which lack endogenous somatostatin receptors. In transfected GH12C1 cells, both SSTR1 and SSTR2 coupled to inhibition of Ca2+ influx and hyperpolarization of membrane potential via a pertussis toxin (PTx)-sensitive mechanism. These effects reflected modulation of ion channel activities which are important for regulation of hormone secretion. Somatostatin analogs MK678 and CH275 acted as subtype selective agonists as expected. In transfected F4C1 cells, both SSTR1 and SSTR2 mediated somatostatin-induced inhibition of adenylyl cyclase via a PTx-sensitive pathway. In addition, activation of SSTR2 in F4C1 cells, but not SSTR1, stimulated phospholipase C (PLC) activity and an increase in [Ca2+]i due to release of Ca2+ from intracellular stores. Unlike adenylyl cyclase inhibition, the PLC-mediated response was only partially sensitive to PTx. To determine the structural determinants in SSTR2 necessary for activation of PLC, we constructed chimeric receptors in which domains of SSTR2 were introduced into SSTR1. Chimeric receptors containing only the third intracellular loop, or all three intracellular loops from SSTR2, mediated inhibition of adenylyl cyclase, but failed to stimulate PLC activity as did wild-type SSTR2. Furthermore, the C-terminal tail of SSTR2 was not required for coupling to PLC. Thus, by expressing individual somatostatin receptor subtypes in pituitary cells, we have identified both overlapping and distinct signaling pathways for SSTR1 and SSTR2, and have shown that sequences other than simply the intracellular domains are required for SSTR2 to couple to the PLC signaling pathway.


Assuntos
Hipófise/metabolismo , Receptores de Somatostatina/metabolismo , Transdução de Sinais , Toxina Adenilato Ciclase , Inibidores de Adenilil Ciclases , Animais , Células CHO , Células COS , Cálcio/metabolismo , Colforsina/farmacologia , Cricetinae , AMP Cíclico/metabolismo , Fosfatos de Inositol/metabolismo , Potenciais da Membrana , Toxina Pertussis , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Células Tumorais Cultivadas , Fosfolipases Tipo C/metabolismo , Fatores de Virulência de Bordetella/farmacologia
19.
J Biol Chem ; 272(4): 2326-33, 1997 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-8999941

RESUMO

To determine whether functional receptor-G protein coupling or signaling are required for internalization of the thyrotropin-releasing hormone receptor (TRHR), we compared the endocytosis of Gq-coupled and uncoupled receptors. A hemagglutinin epitope-tagged TRHR (HA-TRHR) was in the Gq-coupled state when bound to the agonist, MeTRH, and in a nonsignaling state when bound to the HA antibody (12CA5). 12CA5 did not induce an increase in [Ca2+]i or inositol phosphates and did not inhibit [3H]MeTRH binding or MeTRH-induced production of second messengers. Both agonist- and antibody-bound HA-TRHRs were rapidly internalized via the same pathway; internalization was sensitive to hypertonic shock, and both types of internalized receptors were sorted into lysosomes. In addition, the amino acid sequence CNC (positions 335-337) in the C-terminal tail of the TRHR, which is important in ligand-induced receptor internalization as determined by deletion mutagenesis (Nussenzveig, D. R., Heinflink, M., and Gershengorn, M. C. (1993) J. Biol. Chem. 268, 2389-2392), was also important for 12CA5-induced internalization. We expressed two truncated receptors, HA-K338STOP and HA-C335STOP, in GH12C1 pituitary cells. Both HA-TRHR and HA-K338STOP were localized at the plasma membrane of untreated cells and were translocated to intracellular vesicles after MeTRH or 12CA5 binding; however, HA-C335STOP was internalized and recycled constitutively. The intracellular localization of HA-C335STOP was not altered by MeTRH; however, 12CA5 binding induced the disappearance of internalized HA-C335STOP and caused its localization at the plasma membrane, indicating that constitutively cycling HA-C335STOP cannot be reinternalized after antibody binding. Thus, amino acids 335-337, which are important for the internalization of Gq-coupled TRHRs, are also required for the sequestration of functionally uncoupled TRHRs, and in addition, they act as an inhibitory signal that prevents constitutive receptor internalization. Specifically, the Cys residues at positions 335 and 337 are important for preventing constitutive TRHR internalization, because a mutant HA-C335S/C337S receptor was sequestered constitutively. We conclude that release from a negative regulatory internalization sequence or domain is important for HA-TRHR internalization and that the role of the CNC sequence in internalization is independent of functional TRHR-Gq coupling.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Modelos Biológicos , Receptores do Hormônio Liberador da Tireotropina/metabolismo , Transdução de Sinais , Animais , Anticorpos Monoclonais/metabolismo , Células Cultivadas , Técnica Indireta de Fluorescência para Anticorpo , Mutagênese Sítio-Dirigida , Ácido Pirrolidonocarboxílico/análogos & derivados , Ratos , Receptores do Hormônio Liberador da Tireotropina/genética , Relação Estrutura-Atividade , Hormônio Liberador de Tireotropina/análogos & derivados , Hormônio Liberador de Tireotropina/metabolismo , Transferrina/metabolismo
20.
Mol Pharmacol ; 50(6): 1495-501, 1996 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8967970

RESUMO

Capacitative Ca2+ influx, which occurs in response to mobilization of intracellular Ca2+ stores, is a general feature of many cell types. Although the mechanism of capacitative Ca2+ entry is not known, evidence suggests the involvement of small G proteins that are prenylated on a cysteine residue near their carboxyl termini. We have investigated the actions of farnesyl-cysteine analogs on capacitative Ca2+ influx. Using human embryonic kidney 293 cells, we found that S-farnesylthioacetic acid, N-acetyl-S-farnesyl-L-cysteine, N-pivaloyl-S-farnesyl-L-cysteine, and N-acetyl-S-gernylgernyl-L-cysteine blocked the activation of capacitative Ca2+ influx, whereas N-benzoyl-S-farnesyl-S-cysteine had no effect on capacitative Ca2+ entry. Inhibition by S-farnesylthioacetic acid was concentration dependent (5-20 microM) and specific for Ca2+ influx through non-voltage-gated Ca2+ channels. A single protein band of 26-28 kDa was labeled specifically with a photoaffinity analog of farnesylcysteine. GTP binding to the photoaffinity-labeled band was demonstrated. These findings suggest, but do not prove, that a prenylated substrate, possibly a small G protein, is linked functionally to capacitative Ca2+ entry in human embryonic kidney 293 cells.


Assuntos
Cálcio/metabolismo , Cisteína/farmacologia , Marcadores de Afinidade , Linhagem Celular , Cisteína/análogos & derivados , Guanosina Trifosfato/metabolismo , Humanos , Manganês/metabolismo , Ligação Proteica , Tapsigargina/farmacologia
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