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P D Garcia

Publications and source records attributed to P D Garcia.

16 recordsLinked to original sources
PubMed2001

Elevated expression of axin2 and hnkd mRNA provides evidence that Wnt/beta -catenin signaling is activated in human colon tumors.

Genetic studies have identified mutations in key regulators of the Wnt/beta-catenin pathway in a variety of cancers, most frequently in colon cancers. However, whether the pathway is activated in clinical cancer samples is not easily determined, and therefore it is useful to find markers that could be surrogates to show activation of the Wnt/beta-catenin pathway. Gene expression profiles were analyzed in SW620, a colon cancer cell line in which beta-catenin levels are stabilized as a consequence of truncated adenomatous polyposis coli and were compared with profiles of the same cells transfected with antisense oligodeoxynucleotides. Treatment of cells with beta-catenin antisense oligodeoxynucleotides resulted in a decrease in the levels of axin2 and human naked cuticle (hnkd) mRNAs. Interestingly, the proteins encoded by both of these mRNAs are known inhibitors of the beta-catenin pathway. In 30 human cell lines derived from different origins, axin2 and hnkd were expressed only in human colon cancer cell lines that are known to have activating mutations in the Wnt/beta-catenin pathway. Further, levels of both axin2 and hnkd mRNA were also found to be elevated in about 65% of laser microdissected cells from human colon tumors compared with laser microdissected cells of normal morphology from the same patient samples. The increased expression of axin2 and hnkd correlated with truncations in adenomatous polyposis coli in the same patient samples. These results reveal that it is possible to detect activation of a carcinogenic pathway in human cancer samples with specific markers.

Axin Protein↗
PubMed1997

Receptor and betagamma binding sites in the alpha subunit of the retinal G protein transducin.

Transmembrane receptors for hormones, neurotransmitters, light, and odorants mediate their cellular effects by activating heterotrimeric guanine nucleotide-binding proteins (G proteins). Crystal structures have revealed contact surfaces between G protein subunits, but not the surfaces or molecular mechanism through which Galphabetagamma responds to activation by transmembrane receptors. Such a surface was identified from the results of testing 100 mutant alpha subunits of the retinal G protein transducin for their ability to interact with rhodopsin. Sites at which alanine substitutions impaired this interaction mapped to two distinct Galpha surfaces: a betagamma-binding surface and a putative receptor-interacting surface. On the basis of these results a mechanism for receptor-catalyzed exchange of guanosine diphosphate for guanosine triphosphate is proposed.

Aluminum Compounds↗
PubMed1995

Transducin-alpha C-terminal mutations prevent activation by rhodopsin: a new assay using recombinant proteins expressed in cultured cells.

We have measured the activation by recombinant rhodopsin of the alpha-subunit (alpha 1) of retinal transducin (Gt, also recombinant) using a new assay. Cultured cells are transiently transfected with DNAs encoding opsin and the three subunits of Gt (alpha t, beta 1 and gamma 1). In the microsomes of these cells, incubated with 11-cis-retinal, light causes the rapid activation of Gt, as measured by the ability of GTP gamma S to protect alpha t fragments from proteolytic degradation. The activation of Gt is also observed when all-trans-retinal is added to microsomes under constant illumination. Activation depends on both opsin and retinal. Opsin mutants with known defects in activating Gt show similar defects in this assay. alpha t mutations that mimic the corresponding mutations in the alpha-subunit of Gs also produce qualitatively similar effects in this assay. As a first step in a strategy aimed at exploring the relationships between structure and function in the interactions of receptors with G proteins, we tested mutant alpha t proteins with alanine substituted for each of the 10 amino acids at the C-terminus, a region known to be crucial for interactions with rhodopsin. Alanine substitution at four positions moderately (K341) or severely (L344, G348, L349) impairs the susceptibility of alpha 1 to activation by rhodopsin. All four mutants retain their ability to be activated by AIF-4. Two other substitutions (N343 and F350) resulted in very mild defects, while substitutions at the remaining four positions (E342, K345, D346 and C347) had no effect. In combination with previous observations, these results constrain models of the interaction of the C-terminus of alpha t with rhodopsin.

Amino Acid Sequence↗
PubMed1994

Adenovirus-mediated transfection of cultured cells.

We describe here a simple and efficient transfection method for transient expression of cloned genes in cell lines and primary cultured cells. The method involves the use of DEAE-dextran to target DNA to the cellular endocytotic pathway and the use of a human adenovirus to ensure efficient lysis of endosomal vesicles. The procedure allows effective delivery of DNA into the cytoplasm and, therefore, results in a higher fraction of cells expressing exogenous proteins. Using this method, we routinely obtain 60%-90% of COS cells or Chinese hamster ovary cells expressing beta-galactosidase, as determined by in situ staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal). We have also obtained much improved levels of expression in cells that are difficult or impossible to use in transient expression assays, such as rat-1 fibroblasts or primary osteoblast cultures. We successfully used the method to express heteromeric proteins that require subunit assembly for proper function. The method also proved effective to express functions in which the exogenous protein needs to couple to the endogenous cellular machinery. Thus, this transient transfection method should prove valuable for many functional studies in a broad variety of cell lines and primary cultures.

Acetylcholine↗
PubMed1994

Pituitary cell line GH3 expresses two somatostatin receptor subtypes that inhibit adenylyl cyclase: functional expression of rat somatostatin receptor subtypes 1 and 2 in human embryonic kidney 293 cells.

Using a polymerase chain reaction approach, we have studied the expression of somatostatin receptor (SSTR) subtypes in the GH3 rat pituitary cell line, a well established in vitro model for the cellular effects of somatostatin. We found that the previously identified SSTR1 and SSTR2 are the major subtypes expressed in this cell line. No other SSTR subtype was detected by our analysis. Northern blots confirmed that both subtypes, but not SSTR3, are expressed in GH3 cells. We studied the functional expression of both SSTR subtypes by transfection of their cDNAs into human embryonic kidney 293 cells. We found that somatostatin inhibited cAMP accumulation in human embryonic kidney 293 cells only when cells were transfected with either SSTR1 or SSTR2. This inhibition was blocked by treatment of the transfected cells with pertussis toxin, demonstrating that it is mediated by G proteins sensitive to this toxin. In addition, we provide pharmacological evidence that the endogenous SSTR2 subtype mediates inhibition of cAMP accumulation in intact GH3 cells. Our results contradict previous reports that concluded thsat neither SSTR1 nor SSTR2 is involved in inhibition of adenylyl cyclase. The reasons for this apparent contradiction are discussed. We conclude that both SSTR1 and SSTR2 are capable of coupling to pertussis toxin-sensitive G proteins to inhibit adenylyl cyclase.

Adenylate Cyclase Toxin↗
PubMed1992

Identification and molecular cloning of a neuropeptide Y homolog that produces prolonged inhibition in Aplysia neurons.

The neuroendocrine bag cell neurons of the marine mollusk Aplysia produce prolonged inhibition that lasts for more than 2 hr. We purified a peptide from the abdominal ganglion that mimics this inhibition. Mass spectrometry and microsequence analysis indicate that the peptide is 40 aa long and is amidated at its carboxyl terminus. It is highly homologous to vertebrate neuropeptide Y (NPY) and other members of the pancreatic polypeptide family. As determined from cloned cDNA, the gene coding for the precursor protein shares a common structural organization with genes encoding precursors of the vertebrate family. The peptides may therefore have arisen from a common ancestral gene. Bag cell neurons are immunoreactive for Aplysia NPY, and Northern blot analysis indicates that as with its vertebrate counterparts, the peptide is abundantly expressed in the CNS. This suggests that peptides related to NPY may have important functions in the nervous system of Aplysia as well as in other invertebrates.

Amino Acid Sequence↗
PubMed1988

Processing of the native nerve growth factor precursor to form biologically active nerve growth factor.

In the mouse submaxillary gland beta nerve growth factor (beta-NGF) forms a complex with two members of the kallikrein family of serine proteases, termed the alpha- and gamma-subunits of NGF. We demonstrate that the beta-NGF precursor produced in mammalian cells via a recombinant vaccinia virus can be cleaved by stoichiometric quantities of the gamma-subunit to produce beta-NGF. Trypsin in catalytic quantities also produces native beta-NGF. Proper cleavage depends critically on the conformation of the precursor. beta-NGF has at least 10-fold more biological activity than its precursor.

Animals↗
PubMed1988

Heavy-chain binding protein recognizes aberrant polypeptides translocated in vitro.

Immunoglobulin heavy-chain binding protein (BiP, GRP-78) associates tightly in the endoplasmic reticulum (ER) with newly synthesized proteins that are incompletely assembled, have mutant structures, or are incorrectly glycosylated. The function of BiP has been suggested to be to prevent secretion of incorrectly folded or incompletely assembled protein, to promote folding or assembly of proteins, or to solubilize protein aggregates within the ER lumen. Here we examine the interaction of BiP with newly synthesized polypeptides in an in vitro protein translation-translocation system. We find that BiP forms tight complexes with nonglycosylated yeast invertase and incorrectly disulphide-bonded prolactin, but does not associate detectably with either glycosylated invertase or correctly disulphide-bonded prolactin. Moreover, BiP associates detectably only with completed chains of prolactin, not with chains undergoing synthesis. We conclude that BiP recognizes and binds with high affinity in vitro to aberrantly folded or aberrantly glycosylated polypeptides, but not to all nascent chains as they are folding.

Carrier Proteins↗
PubMed1988

Full-length prepro-alpha-factor can be translocated across the mammalian microsomal membrane only if translation has not terminated.

We have previously shown that fully synthesized prepro-alpha-factor (pp alpha F), the precursor for the yeast pheromone alpha-factor, can be translocated posttranslationally across yeast rough microsomal (RM) membranes from a soluble, ribosome-free pool. We show here that this is not the case for translocation of pp alpha F across mammalian RM. Rather we found that a small amount of translocation of full-length pp alpha F is observed, but is solely due to polypeptide chains that were still ribosome bound and covalently attached to tRNA, i.e., not terminated. In addition, both signal recognition particle (SRP) and SRP receptor are required, i.e., the same targeting machinery that is normally responsible for the coupling between protein synthesis and translocation. Thus, the molecular requirements for targeting are distinct from posttranslational translocation across yeast RM. As termination is generally regarded as part of translation, the translocation of full-length pp alpha F across mammalian RM does not occur "posttranslationally," albeit independent of elongation. Most other proteins for which posttranslational translocation across mammalian RM was previously claimed fall into the same category in that ribosome attachment as peptidyl-tRNA is required. To clearly separate these two distinct processes, we suggest that the term posttranslational be reserved for those processes that occur in the complete absence of the translational machinery. We propose the term "ribosome-coupled translocation" for the events described here.

Animals↗
PubMed1988

Targeting of the hepatitis B virus precore protein to the endoplasmic reticulum membrane: after signal peptide cleavage translocation can be aborted and the product released into the cytoplasm.

The major hepatitis B virus (HBV) core protein is a viral structural protein involved in nucleic acid binding. Its coding sequence contains an extension of 29 codons (the "precore" region) at the amino terminus of the protein which is present in a fraction of the viral transcripts. This region is evolutionarily conserved among mammalian and avian HBVs, suggesting it has functional importance, although at least for duck HBV it has been shown to be nonessential for replication of infectious virions. Using in vitro assays for protein translocation across the endoplasmic reticulum membrane, we found that the precore region of the HBV genome encodes a signal sequence. This signal sequence was recognized by signal recognition particle, which targeted the nascent precore protein to the endoplasmic reticulum membrane with efficiencies comparable to those of other mammalian secretory proteins. A 19-amino acid signal peptide was removed by signal peptidase on the lumenal side of the microsomal membrane, generating a protein similar to the HBV major core protein, but containing 10 additional amino acids from the precore region at its amino terminus. Surprisingly, we found that 70-80% of this signal peptidase-cleaved product was localized on the cytoplasmic side of the microsomal vesicles and was not associated with the membranes. We conclude that translocation was aborted by an unknown mechanism, then the protein disengaged from the translocation machinery and was released back into the cytoplasm. Thus, a cytoplasmically disposed protein was created whose amino terminus resulted from signal peptidase cleavage. The remaining 20-30% appeared to be completely translocated into the lumen of the microsomes. A deletion mutant lacking the carboxy-terminal nucleic acid binding domain of the precore protein was similarly partitioned between the lumen of the microsomes and the cytoplasmic compartment, indicating that this highly charged domain is not responsible for the aborted translocation. We discuss the implications of our findings for the protein translocation process and suggest a possible role in the virus life cycle.

Cytoplasm↗
PubMed1987

Wild type and mutant signal peptides of Escherichia coli outer membrane lipoprotein interact with equal efficiency with mammalian signal recognition particle.

The signal peptide of the outer membrane lipoprotein (OMLP) of Escherichia coli was shown to be capable of promoting protein translocation across mammalian microsomal membranes in vitro. We assayed translocation of a fusion protein containing the OMLP signal peptide and nine amino acids of OMLP fused in frame to beta-lactamase. The efficiency with which the mammalian translocation machinery recognizes and accepts the OMLP signal peptide as substrate is indistinguishable from that of mammalian secretory proteins. Upon translocation mammalian signal peptidase processes the pre-OMLP-beta-lactamase protein at different sites than are utilized in vivo by E. coli OMLP signal peptidase (signal peptidase II) but that can be predicted as mammalian signal peptidase cleavage sites. Mutants in the OMLP signal peptide were tested for their ability to promote translocation of the fusion protein in this assay system. It has been shown previously that mutants in the positively charged amino acids at the amino terminus of the signal peptide severely delay the translocation of OMLP in vivo in E. coli. However, these mutants had no detectable effect either on signal recognition by mammalian signal recognition particle or on the efficiency of translocation itself.

Amino Acid Sequence↗
PubMed1986

In vitro protein translocation across the yeast endoplasmic reticulum: ATP-dependent posttranslational translocation of the prepro-alpha-factor.

The in vitro synthesized precursor of the alpha-factor pheromone, prepro-alpha-factor, of Saccharomyces cerevisiae was translocated across yeast microsomal membranes in either a homologous or a wheat germ cell free system. Translocated prepro-alpha-factor was glycosylated, sedimented with yeast microsomal vesicles, and was protected from digestion by added protease, but was soluble after alkaline sodium carbonate treatment. Thus prepro-alpha-factor was properly sequestered within yeast microsomal vesicles, but was not integrated into the lipid bilayer. In marked contrast to protein translocation across mammalian microsomal membranes, translocation of prepro-alpha-factor across yeast microsomal membranes could occur posttranslationally. This reaction required protein components in the yeast microsomal fraction that could be inactivated by alkylation or proteolysis, was ATP-dependent, and was insensitive to the presence of a variety of uncouplers and ionophores.

Adenosine Triphosphate↗
PubMed1986

Integration of hepatitis B virus DNA in chromosome-specific satellite sequences.

We previously reported the cloning and detailed analysis of the integrated hepatitis B virus sequences in a human hepatoma cell line. We report here the integration of at least one of hepatitis B virus at human satellite DNA sequences. The majority of the cellular sequences identified by this satellite DNA were organized as a multimeric composition of a 0.6-kilobase EcoRI fragment. This clone hybridized in situ almost exclusively to the centromeric heterochromatin of chromosomes 1 and 16 and to a lower extent to chromosome 2 and to the heterochromatic region of the Y chromosome. The immediate flanking host sequence appeared as a hierarchy of repeating units which were almost identical to a previously reported human satellite III DNA sequence.

Carcinoma, Hepatocellular↗
PubMed1984

Cloning and analysis of integrated hepatitis virus sequences from a human hepatoma cell line.

We report here the isolation by molecular cloning and the analysis by heteroduplex and restriction enzyme mapping of seven distinct DNA fragments containing hepatitis B virus (HBV) sequences from genomic DNA of the PLC/PRF/5 human liver carcinoma cell line (the Alexander cell). No intact full-length HBV genomes were present. Three different patterns of organization of HBV fragments were detected. These included two linear fragments without detectable rearrangement, three other HBV fragments with internal deletions, and two HBV fragments containing long inverted duplications. HBsAg sequences are preferentially included in the integrated fragment, whereas the core gene is preferentially eliminated. Several of the integrated HBV fragments might act as templates for the synthesis of functional HBsAg mRNA, whereas only one clone could produce a full core antigen transcript.

Base Sequence↗
PubMedDate not supplied

Topology of signal recognition particle receptor in endoplasmic reticulum membrane.

The signal recognition particle (SRP) receptor is an integral membrane protein of the endoplasmic reticulum which, in conjunction with SRP, ensures the correct targeting of nascent secretory proteins to this membrane system. From the complementary DNA sequence we have deduced the complete primary structure of the SRP receptor and established that its amino-terminal region is anchored in the membrane. The anchor fragment and the cytoplasmic fragment contribute jointly to a functionally important region which is highly charged and may function in nucleic acid binding.

Amino Acid Sequence↗