PubMed Health⌕ Search

Biomedical subjects

H G Dohlman

Publications and source records attributed to H G Dohlman.

At least 37 records · Page 2Linked to original sources
PubMed1996

Sst2, a negative regulator of pheromone signaling in the yeast Saccharomyces cerevisiae: expression, localization, and genetic interaction and physical association with Gpa1 (the G-protein alpha subunit).

Sst2 is the prototype for the newly recognized RGS (for regulators of G-protein signaling) family. Cells lacking the pheromone-inducible SST2 gene product fail to resume growth after exposure to pheromone. Conversely, overproduction of Sst2 markedly enhanced the rate of recovery from pheromone-induced arrest in the long-term halo bioassay and detectably dampened signaling in a short-term assay of pheromone response (phosphorylation of Ste4, Gbeta subunit). When the GPA1 gene product (Galpha subunit) is absent, the pheromone response pathway is constitutively active and, consequently, growth ceases. Despite sustained induction of Sst2 (observed with specific anti-Sst2 antibodies), gpa1delta mutants remain growth arrested, indicating that the action of Sst2 requires the presence of Gpa1. The N-terminal domain (residues 3 to 307) of Sst2 (698 residues) has sequence similarity to the catalytic regions of bovine GTPase-activating protein and human neurofibromatosis tumor suppressor protein; segments in the C-terminal domain of Sst2 (between residues 417 and 685) are homologous to other RGS proteins. Both the N- and C-terminal domains were required for Sst2 function in vivo. Consistent with a role for Sst2 in binding to and affecting the activity of Gpa1, the majority of Sst2 was membrane associated and colocalized with Gpa1 at the plasma membrane, as judged by sucrose density gradient fractionation. Moreover, from cell extracts, Sst2 could be isolated in a complex with Gpa1 (expressed as a glutathione S-transferase fusion); this association withstood the detergent and salt conditions required for extraction of these proteins from cell membranes. Also, SST2+ cells expressing a GTPase-defective GPA1 mutant displayed an increased sensitivity to pheromone, whereas sst2 cells did not. These results demonstrate that Sst2 and Gpa1 interact physically and suggest that Sst2 is a direct negative regulator of Gpa1.

Amino Acid Sequence↗
PubMed1995

Inhibition of G-protein signaling by dominant gain-of-function mutations in Sst2p, a pheromone desensitization factor in Saccharomyces cerevisiae.

Genetic analysis of cell-cell signaling in Saccharomyces cerevisiae has led to the identification of a novel factor, known as Sst2p, that promotes recovery after pheromone-induced growth arrest (R. K. Chan and C. A. Otte, Mol. Cell. Biol. 2:11-20, 1982). Loss-of-function mutations lead to increased pheromone sensitivity, but this phenotype is partially suppressed by overexpression of the G protein alpha subunit gene (GPA1). Suppression is allele specific, however, suggesting that there is direct interaction between the two gene products. To test this model directly, we isolated and characterized several dominant gain-of-function mutants of SST2. These mutations block the normal pheromone response, including a loss of pheromone-stimulated gene transcription, cell cycle growth arrest, and G protein myristoylation. Although the SST2 mutations confer a pheromone-resistant phenotype, they do not prevent downstream activation by overexpression of G beta (STE4), a constitutively active G beta mutation (STE4Hpl), or a disruption of GPA1. None of the SST2 alleles affects the expression or stability of G alpha. These results point to the G protein alpha subunit as being the direct target of Sst2p action and underscore the importance of this novel desensitization factor in G-protein-mediated signaling.

Cell Division↗
PubMed1993

Pheromone action regulates G-protein alpha-subunit myristoylation in the yeast Saccharomyces cerevisiae.

Myristic acid (C14:0) is added to the N-terminal glycine residue of the alpha subunits of certain receptor-coupled guanine nucleotide-binding regulatory proteins (G proteins). The G alpha subunit (GPA1 gene product) coupled to yeast pheromone receptors exists as a pool of both myristoylated and unmyristolyated species. After treatment of MATa cells with alpha factor, the myristoylated form of Gpa1p increases dramatically, and the unmyristoylated form decreases concomitantly. This pheromone-stimulated shift depends on the function of STE2 (alpha-factor receptor), STE11 (a protein kinase in the response pathway), and NMT1 (myristoyl-CoA:protein N-myristoyltransferase) genes and uses the existing pool of fatty acids (is not blocked by cerulenin). Myristoylated Gpa1p persists long after pheromone is removed. Because myristoylation is essential for proper G alpha-G beta gamma association and receptor coupling, pheromone-dependent stimulation of Gpa1p myristoylation may be an important contributing factor in adaptation after signal transmission.

Amino Acid Sequence↗
PubMed1993

The a-factor transporter (STE6 gene product) and cell polarity in the yeast Saccharomyces cerevisiae.

STE6 gene product is required for secretion of the lipopeptide mating pheromone a-factor by Saccharomyces cerevisiae MATa cells. Radiolabeling and immunoprecipitation, either with specific polyclonal antibodies raised against a TrpE-Ste6 fusion protein or with mAbs that recognize c-myc epitopes in fully functional epitope-tagged Ste6 derivatives, demonstrated that Ste6 is a 145-kD phosphoprotein. Subcellular fractionation, various extraction procedures, and immunoblotting showed that Ste6 is an intrinsic plasma membrane-associated protein. The apparent molecular weight of Ste6 was unaffected by tunicamycin treatment, and the radiolabeled protein did not bind to concanavalin A, indicating that Ste6 is not glycosylated and that glycosylation is not required either for its membrane delivery or its function. The amino acid sequence of Ste6 predicts two ATP-binding folds; correspondingly, Ste6 was photoaffinity-labeled specifically with 8-azido-[alpha-32P]ATP. Indirect immunofluorescence revealed that in exponentially growing MATa cells, the majority of Ste6 showed a patchy distribution within the plasma membrane, but a significant fraction was found concentrated in a number of vesicle-like bodies subtending the plasma membrane. In contrast, in MATa cells exposed to the mating pheromone alpha-factor, which markedly induced Ste6 production, the majority of Ste6 was incorporated into the plasma membrane within the growing tip of the elongating cells. The highly localized insertion of this transporter may establish pronounced anisotropy in a-factor secretion from the MATa cell, and thereby may contribute to the establishment of the cell polarity which restricts partner selection and cell fusion during mating to one MAT alpha cell.

ATP-Binding Cassette Transporters↗
PubMed1990

Control of yeast mating signal transduction by a mammalian beta 2-adrenergic receptor and Gs alpha subunit.

To facilitate functional and mechanistic studies of receptor-G protein interactions, [corrected] the human beta 2-adrenergic receptor (h beta-AR) has been expressed in Saccharomyces cerevisiae. This was achieved by placing a modified h beta-AR gene under control of the galactose-inducible GAL1 promoter. After induction by galactose, functional h beta-AR was expressed at a concentration several hundred times as great as that found in any human tissue. As determined from competitive ligand binding experiments, h beta-AR expressed in yeast displayed characteristic affinities, specificity, and stereoselectivity. Partial activation of the yeast pheromone response pathway by beta-adrenergic receptor agonists was achieved in cells coexpressing h beta-AR and a mammalian G protein (Gs) alpha subunit-demonstrating that these components can couple to each other and to downstream effectors when expressed in yeast. This in vivo reconstitution system provides a new approach for examining ligand binding and G protein coupling to cell surface receptors.

Amino Acid Sequence↗
PubMed1990

Role of extracellular disulfide-bonded cysteines in the ligand binding function of the beta 2-adrenergic receptor.

Evidence is presented for a role of disulfide bridging in forming the ligand binding site of the beta 2-adrenergic receptor (beta AR). The presence of disulfide bonds at the ligand binding site is indicated by "competitive" inhibition by dithiothreitol (DTT) in radioligand binding assays, by specific protection by beta-adrenergic ligands of these effects, and by the requirement of disulfide reduction for limit proteolysis of affinity ligand labeled receptor. The kinetics of binding inhibition by DTT suggest at least two pairs of disulfide-bonded cysteines essential for normal binding. Through site-directed mutagenesis, we indeed were able to identify four cysteines which are critical for normal ligand binding affinities and for the proper expression of functional beta AR at the cell surface. Unexpectedly, the four cysteines required for normal ligand binding are not those located within the hydrophobic transmembrane domains of the receptor (where ligand binding is presumed to occur) but lie in the extracellular hydrophilic loops connecting these transmembrane segments. These findings indicate that, in addition to the well-documented involvement of the membrane-spanning domains of the receptor in ligand binding, there is an important and previously unsuspected role of the hydrophilic extracellular domains in forming the ligand binding site.

Animals↗
PubMed1990

Synthetic peptides of the hamster beta 2-adrenoceptor as substrates and inhibitors of the beta-adrenoceptor kinase.

1. The beta-adrenoceptor is one of a number of G protein-coupled receptors which have been proposed to contain seven transmembrane alpha-helices. The function of this receptor appears to be regulated by phosphorylation by a specific enzyme, the beta-adrenoceptor kinase. Synthetic peptides which comprise each of the proposed intra- and extracellular domains of the beta 2-adrenoceptor have been tested as potential substrates and inhibitors of the beta-adrenoceptor kinase. 2. Two peptides which encompass the middle and terminal portions of the carboxyl tail of the receptor served as substrates by beta-adrenoceptor kinase. The kinetics of the phosphorylation reaction, however, suggest that these peptides are 10(6)-fold poorer substrate than the agonist occupied receptor. 3. A number of synthetic peptides also served as inhibitors of beta 2-adrenoceptor phosphorylation by beta-adrenoceptor kinase. In particular, a peptide which comprised the first intracellular loop of the beta 2-adrenoceptor (amino acids 56-74) inhibited most effectively with an IC50 of 40 microM. 4. These results suggest that multiple intracellular regions of the beta-receptor may serve as potential sites of interaction with beta-adrenoceptor kinase. Moreover, these regions may serve as potential targets for the development of specific inhibitors of beta-adrenoceptor kinase which could be used to block homologous desensitization.

Amino Acid Sequence↗
PubMed1988

Identification and sequence of a binding site peptide of the beta 2-adrenergic receptor.

p-(Bromoacetamido)benzyl-1-[125I]iodocarazolol (125I-pBABC) is a potent derivative of the beta-adrenergic receptor antagonist p-aminobenzylcarazolol. Treatment of the receptor with 125I-pBABC results in efficient covalent incorporation of the ligand into the receptor binding site. Extensive degradation of 125I-pBABC-labeled beta 2-adrenergic receptor with either cyanogen bromide or Staphylococcus aureus V8 protease results in specifically labeled fragments having Mr's of about 1600 and 3500, respectively. Because the primary structure of the beta 2-adrenergic receptor is known, and these proteolytic reagents are highly sequence specific, the site of 125I-pBABC incorporation may be deduced from the sizes of the specifically labeled fragments. Thus the fragment generated by cyanogen bromide cleavage corresponds to residues 83-96, a region of 14 amino acids included in the second membrane spanning domain (helix II) of the beta 2-adrenergic receptor. This assignment was confirmed by direct amino acid sequencing of this labeled fragment, though the actual amino acid modified could not be determined. These data permit the assignment of a part of the hormone binding region of the beta 2-adrenergic receptor.

Amino Acid Sequence↗
PubMed1987

The multiple membrane spanning topography of the beta 2-adrenergic receptor. Localization of the sites of binding, glycosylation, and regulatory phosphorylation by limited proteolysis.

The beta 2-adrenergic receptor (beta-AR) is an integral membrane glycoprotein of apparent Mr approximately equal to 64,000. The amino acid sequence deduced from the beta-AR gene reveals homology with the visual pigment rhodopsin of retinal rod outer segments. We have proposed a structural model of beta-AR which is similar to that elucidated for rhodopsin. In this paper we identify a number of structural and topographical characteristics of beta-AR consistent with the model through the use of limited proteolysis. Limited trypsinization of beta-AR reconstituted in lipid vesicles yields two insoluble (integral membrane) domains of Mr approximately equal to 38,000 and 26,000. Identical results were obtained in intact cells, indicating that the cleavage site of the receptor is accessible at the extracellular surface of the plasma membrane. The amino-terminal domain (38 kDa) contains the ligand binding site (as revealed by photoaffinity labeling) and the sites of glycosylation (as revealed by its sensitivity to endoglycosidase F), whereas the carboxyl-terminal domain (26 kDa) contains all the sites of in vitro phosphorylation by cAMP-dependent protein kinase and the beta-adrenergic receptor kinase. Of four canonical sites for N-linked glycosylation, two near the amino and two near the carboxyl terminus, only those in the amino-terminal domain (Asn6 and Asn15) are utilized and sensitive to endoglycosidase F. Carboxypeptidase Y treatment of reconstituted native beta-adrenergic receptor generates a truncated (approximately 57 kDa) glycopeptide that has lost most of the sites phosphorylated by beta-AR kinase and one of the sites phosphorylated by protein kinase A. The various features delineated, including the length of the carboxypeptidase Y-sensitive region, the extracellular location of the trypsin-sensitive site, the location of the sites of phosphorylation and glycosylation all constrain the receptor to a rhodopsin-like structure with multiple membrane spanning segments.

Amino Acid Sequence↗
PubMed1987

Delineation of the intronless nature of the genes for the human and hamster beta 2-adrenergic receptor and their putative promoter regions.

The beta 2-adrenergic receptor is the first adenylate cyclase-coupled receptor to be cloned. We provide here a detailed characterization of its complete gene in both the human and hamster which reveals several unusual and provocative features. The genes are present in a single copy, are intronless, and are bounded by homologous 18-bp (base pair) direct repeats. These findings suggest that the beta 2-adrenergic receptor may have arisen as a processed gene for another related gene. Genomic Southern blots done at reduced stringency in fact reveal additional weak signals. The human and hamster gene sequences 5' to the principal site of transcription initiation are highly homologous and share many characteristics of promoters for housekeeping genes. Moreover, there is present in the human genome a long (777 bp) open reading frame which is in frame with the beta-adrenergic receptor coding block and which ends only 234 bp 5' to the initiator methionine of the receptor. An unusual cDNA has been found, transcribed from a putative second more 5' promoter which contains the 5' half of the beta-adrenergic receptor as well as 1065-bp 5' to the receptor coding region, including the entire upstream long open reading frame (sufficient to encode a putative protein of Mr approximately 28,000).

Animals↗
PubMed1987

cDNA for the human beta 2-adrenergic receptor: a protein with multiple membrane-spanning domains and encoded by a gene whose chromosomal location is shared with that of the receptor for platelet-derived growth factor.

We have isolated and sequenced a cDNA encoding the human beta 2-adrenergic receptor. The deduced amino acid sequence (413 residues) is that of a protein containing seven clusters of hydrophobic amino acids suggestive of membrane-spanning domains. While the protein is 87% identical overall with the previously cloned hamster beta 2-adrenergic receptor, the most highly conserved regions are the putative transmembrane helices (95% identical) and cytoplasmic loops (93% identical), suggesting that these regions of the molecule harbor important functional domains. Several of the transmembrane helices also share lesser degrees of identity with comparable regions of select members of the opsin family of visual pigments. We have localized the gene for the beta 2-adrenergic receptor to q31-q32 on chromosome 5. This is the same position recently determined for the gene encoding the receptor for platelet-derived growth factor and is adjacent to that for the FMS protooncogene, which encodes the receptor for the macrophage colony-stimulating factor.

Amino Acid Sequence↗
PubMed1982

Metastasis in a rabbit choroidal melanoma model.

A substrain of Greene hamster melanoma adapted to grow in the choroid of the rabbit eye is shown to have the tendency to metastasize. Twenty-one normal rabbits were studied for the effects of vascular inoculation of a tumor cell suspension prepared from this choroidal melanoma, and two rabbits were used for long-term observation of the natural untreated course of the choroidal melanoma. In both groups, metastases to the liver, lung, kidney, and other organs were demonstrated.

Animals↗
PubMed1982

Induction of ocular neoplasms in Fischer rats by intraocular injection of nickel subsulfide.

Nickel subsulfide, alpha Ni3S2, was administered to albino Fischer rats by a single injection into the vitreous body of the right eye (0.5 mg alpha Ni3S2/rat, suspended in 20 microliter of NaCl vehicle). Control rats received a similar injection of the vehicle. Malignant tumors developed in the injected eyes of 14/15 alpha Ni3S2-treated rats by 8 months (vs. 0/11 controls, p less than 0.001). Five of the injected eyes of alpha Ni3S2-treated rats contained multiple tumors. The 21 eye tumors that were induced by alpha Ni3S2 included 11 melanomas, four retinoblastomas, three gliomas, and three unclassified malignant neoplasms. Three of the melanomas developed extraocular extensions; one of the melanomas metastasized to lungs and brain. Although the melanomas arose from amelanotic uveal melanocytes, melanosomes were observed in electron micrographs of the tumor cells. This study provides a new experimental model for chemical induction of ocular neoplasms. As a procedure to test the carcinogenicity of nickel compounds, intraocular injection has the advantages of short latency period, high tumor incidence, and ease of tumor detection.

Animals↗