Alpha-factor Receptor Research Articles

Efficient signal transduction by a chimeric yeast-mammalian G protein alpha subunit Gpa1-Gsalpha covalently fused to the yeast receptor Ste2.

Saccharomyces cerevisiae uses G protein-coupled receptors for signal transduction. We show that a fusion protein between the alpha-factor receptor (Ste2) and the Galpha subunit (Gpa1) transduces the signal efficiently in yeast cells devoid of the endogeneous STE2 and GPA1 genes. To evaluate the function of different domains of Galpha, a chimera between the N-terminal region of yeast Gpa1 and the C-terminal region of rat Gsalpha has been constructed. This chimeric Gpa1-Gsalpha is capable of restoring viability to haploid gpa1Delta cells, but signal transduction is prevented. This is consistent with evidence showing that the C-terminus of the homologous Galpha is required for receptor-G protein recognition. Surprisingly, a fusion protein between Ste2 and Gpa1-Gsalpha is able to transduce the signal efficiently. It appears, therefore, that the C-terminus of Galpha is mainly responsible for bringing the G protein into the close proximity of the receptor's intracellular domains, thus ensuring efficient coupling, rather than having a particular role in transmitting the signal. To confirm this conclusion, we show that two proteins interacting with each other (such as Snf1 and Snf4, or Ras and Raf), each of them fused either to the receptor or to the chimeric Galpha, allow efficient signal transduction.

Ubiquitin-dependent internalization and down-regulation of plasma membrane proteins.

The modification of cytosolic proteins with polyubiquitin chains targets them for recognition and degradation by the multisubunit proteolytic particle, the 26S proteasome. Membrane proteins are also substrates for ubiquitination. Integral membrane proteins of the endoplasmic reticulum are ubiquitinated and destroyed by the proteasome. However, it has been shown recently that the ubiquitination of Saccharomyces cerevisiae plasma membrane proteins signals their degradation by the proteolytic system in the lysosome-like vacuole. Ubiquitination of several different classes of cell surface proteins serves as a signal for their entry into the endocytic pathway; this leads to their transport to the vacuole, where they are permanently inactivated by degradation. In yeast, ubiquitin has been implicated as an internalization signal for most, if not all, endogenous plasma membrane proteins that are known to be endocytosed. Ubiquitin-dependent internalization has been best characterized for two proteins: the mating pheromone alpha-factor receptor and the uracil permease. Some mammalian cell surface receptors are also ubiquitinated at the plasma membrane. Ubiquitination machinery is required for ligand-induced endocytosis of the growth hormone receptor, suggesting that ubiquitin-dependent endocytosis and sorting is also an important regulatory process in mammalian cells. Mammalian receptors may also be down-regulated through the degradation of their cytosolic domains by a proteasome-dependent pathway.

Position one analogs of the Saccharomyces cerevisiae tridecapeptide pheromone.

Analogs of the Saccharomyces cerevisiae alpha-mating factor [WHWLQLKPGQPMY], in which a variety of residues replaced Trp1 were synthesized and assayed for biological activity and receptor affinity. Analogs containing Gly or Leu or many different aromatic residues in position 1 of the peptide exhibited bioactivity in a growth arrest assay slightly greater than, or equal to, that of the parent pheromone, whereas the Glu1 and Lys1 analogs exhibited significantly lower bioactivity. Analogs with an aromatic replacement at position 1 had 3- to 6-fold lower receptor affinity than the parent peptide, whereas analogs with a hydrophilic residue at the N-terminus exhibited large reductions in receptor affinity with the peptide with Glu in position 1 showing a 120-fold reduction. N alpha-Acetylation had little effect on bioactivity but lowered receptor affinity by 20- to 40-fold. Amidation of the carboxyl terminus resulted in a 10-fold decrease in activity and a 160-fold decrease in receptor affinity. These results indicate that the alpha-factor receptor has a large hydrophobic binding pocket, possibly containing a negatively charged side-chain, which interacts with the N-terminus of alpha-factor. The lack of correlation between activity and binding and several analogs suggests that small residues near the N-terminus of alpha-factor may be very efficient in triggering isomerization of the receptor to its activated state in the first step of the signal transduction pathway.

In vitro pancreatic ductal cell carcinogenesis.

Our experiments were designed to identify initial biochemical and biological changes that occur during pancreatic carcinogenesis. TAKA-1, an immortal hamster pancreatic ductal cell line, was treated in vitro for up to 11 weeks with the pancreatic carcinogen N-nitorosobis(2-oxopropyl)amine (BOP). These treated cells were designated TAKA-1 + BOP. The growth of TAKA-1 and TAKA-1 + BOP cell lines was investigated in soft agar and in hamsters intradermally. The resulting tumor from TAKA-1 + BOP was re-cultured in vitro and designated TAKA-1 + BOP-T. Mutation of c-K-ras and p53 oncogenes, chromosomal changes, expression of transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) receptor and several biochemical markers were examined in all cell lines. TAKA-1 + BOP but not TAKA-1 cells grew in soft agar and produced an invasive tumor in vivo. However, there were no differences in cell growth rate, DNA flow cytometry, or immunohistochemical findings between the non-transformed and transformed cells. TAKA-1, TAKA-1 + BOP and TAKA-1 + BOP-T cells all expressed mRNA of TGF-alpha and EGF receptor in a comparable pattern. DNA sequence analysis following polymerase chain reaction showed that neither TAKA-1 nor TAKA-1 + BOP cells has a mutation of c-K-ras or p53. Karyotype analysis demonstrated that TAKA-1 + BOP cells had more chromosomal abnormalities compared with TAKA-1 cells. Mutation of c-K-ras and p53 was not essential for carcinogenesis in hamster pancreatic ductal cells in vitro. In conclusion, immortality of the TAKA-1 cells caused expression of TGF-alpha to the same extent as in malignant cells. Chromosomal and ultrastructural patterns were the only differences detected between the non-transformed and BOP-transformed cells.

Linkage of the ubiquitin-conjugating system and the endocytic pathway in ligand-induced internalization of the growth hormone receptor

The major function of the ubiquitin-conjugating system is the targeting of cytosolic and nuclear proteins for degradation by the proteasome. Recently, ubiquitin conjugation has been implicated in the downregulation of signalling receptors such as the mammalian growth hormone receptor (GHR) and the alpha-factor receptor in yeast. By examining truncated receptors, the internalization-deficient receptor mutant F327A and conditions under which clathrin-mediated GHR endocytosis is inhibited, we show here that GHR ubiquitination and ligand-induced GHR internalization are coupled events. Previously, we had shown that GHR endocytosis is dependent on an intact ubiquitination system. Here we present evidence that GHR ubiquitination depends on an intact endocytic pathway. Our data indicate that the ubiquitin-conjugating system and the endocytic pathway interact at the cytoplasmic tail of the GHR at the plasma membrane, where they cooperate to regulate internalization of the GHR.

Expression of transforming growth factor alpha and epidermal growth factor receptor in human hepatocellular carcinoma.

Transforming growth factor alpha (TGF-alpha) is thought to be involved in liver regeneration, cellular proliferation, and hepatocarcinogenesis. We have looked at the relationship between TGF-alpha and it's receptor, and have attempted to relate the expression of TGF-alpha and it's receptor to the differentiation of hepatocellular carcinoma (HCC) on serial sections of HCC. We examined immunohistochemically the expression of the TGF-alpha and of epidermal growth factor receptor (EGFR) proteins in the same area of 53 nodules (< 5 cm in diameter) of HCC obtained from patients. Immunoreactive proteins were visualized by using a biotin-streptoavidin system (LSAB Kil, Dako). TGF-alpha was strongly expressed in 29 of 53 (54.7%) nodules. Specimens strongly positive for TGF-alpha were found mainly in well-differentiated HCC, while specimens positive for EGFR were found mainly in poorly differentiated HCC (p < 0.05). In the tissues that stained weakly positive for TGF-alpha, the expression of EGFR differed significantly, according to the degree of HCC histologic differentiation (p < 0.05). These results led us to speculate that the expression of TGF-alpha and EGFR might be related to the pattern of histologic differentiation of HCC.

The ankyrin repeat-containing protein Akr1p is required for the endocytosis of yeast pheromone receptors.

The Saccharomyces cerevisiae a-factor receptor (Ste3p) requires its C-terminal cytoplasmic tail for endocytosis. Wild-type receptor is delivered to the cell surface via the secretory pathway but remains there only briefly before being internalized and delivered to the vacuole for degradation. Receptors lacking all or part of the cytoplasmic tail are not subject to this constitutive endocytosis. We used the cytoplasmic tail of Ste3p as bait in the two-hybrid system in an effort to identify other proteins involved in endocytosis. One protein identified was Akr1p, an ankyrin repeat-containing protein. We applied three criteria to demonstrate that Akr1p is involved in the constitutive endocytosis of Ste3p. First, when receptor synthesis is shut off, akr1 delta cells retain the ability to mate longer than do AKR1 cells. Second, Ste3p half-life is increased by greater than 5-fold in akr1 delta cells compared with AKR1 cells. Third, after a pulse of synthesis, newly synthesized receptor remains at the cell surface in akr1 delta mutants, whereas it is rapidly internalized in AKR1 cells. Specifically, in akr1 delta mutants, newly synthesized receptor is accessible to exogenous protease, and by indirect immunofluorescence, the receptor is located at the cell surface. akr1 delta cells are also defective for endocytosis of the alpha-factor receptor (Ste2p). Despite the block to constitutive endocytosis exhibited by akr1 delta cells, they are competent to carry out ligand-mediated endocytosis of Ste3p. In contrast, akr1 delta cells cannot carry out ligand-mediated endocytosis of Ste2p. We discuss the implications for Akr1p function in endocytosis and suggest a link to the regulation of ADP-ribosylation proteins (Arf proteins).

Expression and Purification of the Saccharomyces cerevisiae α-Factor Receptor (Ste2p), a 7-Transmembrane-segment G Protein-coupled Receptor

A plasmid vector was developed that permitted high-level expression of a functional form of the Saccharomyces cerevisiae alpha-factor receptor (the STE2 gene product) tagged at its C-terminal end with an epitope (FLAG) and a His6 tract. When expressed in yeast from this plasmid, Ste2p was produced at a level at least 3-fold higher than that reported previously for any other 7-transmembrane-segment receptor expressed in the same cells. For purification, isolated cell membranes containing the overexpressed receptor were solubilized with detergent under specific conditions and subjected to immobilized metal affinity chromatography. Yields as high as 1 mg of nearly homogeneous (95%) receptor were routinely obtained even from relatively small scale preparations (60 g of frozen cell paste). The purified receptor was reconstituted into artificial phospholipid vesicles. Radioligand binding studies demonstrated that the purified receptor, in the reconstituted vesicles, bound its tridecapeptide ligand (alpha-factor) with a KD (155 nM) consistent with the affinity expected for this receptor in the absence of its associated G protein. Efficient restoration of ligand binding activity upon reconstitution required the addition of solubilized membranes prepared from a yeast strain lacking the receptor. Sufficient amounts of active material can be obtained by this procedure to allow physical studies of this receptor and other 7-transmembrane-segment receptors expressed in this system.

Analysis of the receptor binding domain of Gpa1p, the G(alpha) subunit involved in the yeast pheromone response pathway.

The Saccharomyces cerevisiae G protein alpha subunit Gpa1p is involved in the response of both MATa and MAT alpha cells to pheromone. We mutagenized the GPA1 C terminus to characterize the receptor-interacting domain and to investigate the specificity of the interactions with the a- and alpha-factor receptors. The results are discussed with respect to a structural model of the Gpa1p C terminus that was based on the crystal structure of bovine transducin. Some mutants showed phenotypes different than the pheromone response and mating defects expected for mutations that affect receptor interactions, and therefore the mutations may affect other aspects of Gpa1p function. Most of the mutations that resulted in pheromone response and mating defects had similar effects in MATa and MAT alpha cells, suggesting that they affect the interactions with both receptors. Overexpression of the pheromone receptors increased the mating of some of the mutants tested but not the wild-type strain, consistent with defects in mutant Gpa1p-receptor interactions. The regions identified by the mating-defective mutants correlated well with the regions of mammalian G(alpha) subunits implicated in receptor interactions. The strongest mating type-specific effects were seen for mutations to proline and a mutation of a glycine residue predicted to form a C-terminal beta turn. The analogous beta turn in mammalian G(alpha) subunits undergoes a conformational change upon receptor interaction. We propose that the conformation of this region of Gpa1p differs during the interactions with the a- and alpha-factor receptors and that these mating type-specific mutations preclude the orientation necessary for interaction with one of the two receptors.

A C-terminal di-leucine motif and nearby sequences are required for NH4(+)-induced inactivation and degradation of the general amino acid permease, Gap1p, of Saccharomyces cerevisiae.

The general amino acid permease, Gap1, of Saccharomyces cerevisiae is very active in cells grown on proline as the sole nitrogen source. Adding NH4+ to the medium triggers inactivation and degradation of the permease via a regulatory process involving Npi1p/Rsp5p, a ubiquitin-protein ligase. In this study, we describe several mutations affecting the C-terminal region of Gap1p that render the permease resistant to NH4(+)-induced inactivation. An in vivo isolated mutation (gap1pgr) causes a single Glu-->Lys substitution in an amino acid context similar to the DXKSS sequence involved in ubiquitination and endocytosis of the yeast alpha-factor receptor, Ste2p. Another replacement, substitution of two alanines for a di-leucine motif, likewise protects the Gap1 permease against NH4(+)-induced inactivation. In mammalian cells, such a motif is involved in the internalization of several cell-surface proteins. These data provide the first indication that a di-leucine motif influences the function of a plasma membrane protein in yeast. Mutagenesis of a putative phosphorylation site upstream from the di-leucine motif altered neither the activity nor the regulation of the permease. In contrast, deletion of the last eleven amino acids of Gap1p, a region conserved in other amino acid permeases, conferred resistance to NH4+ inactivation. Although the C-terminal region of Gap1p plays an important role in nitrogen control of activity, it was not sufficient to confer this regulation to two NH4(+)-insensitive permeases, namely the arginine (Can1p) and uracil (Fur4p) permeases.

The linker region of the ABC-transporter Ste6 mediates ubiquitination and fast turnover of the protein.

Upon block of endocytosis, the a-factor transporter Ste6 accumulates in a ubiquitinated form at the plasma membrane. Here we show that the linker region, which connects the two homologous halves of Ste6, contains a signal which mediates ubiquitination and fast turnover of Ste6. This signal was also functional in the context of another plasma membrane protein. Deletion of an acidic stretch in the linker region ('A-box') strongly stabilized Ste6. The A-box contains a sequence motif ('DAKTI') which resembles the putative endocytosis signal of the alpha-factor receptor Ste2 ('DAKSS'). Deletion of the DAKTI sequence also stabilized Ste6 but, however, not as strongly as the A-box deletion. There was a correlation between the half-life of the mutants and the degree of ubiquitination: while ubiquitination of the deltaDAKTI mutant was reduced compared with wild-type Ste6, no ubiquitination could be detected for the more stable deltaA-box variant. Loss of ubiquitination seemed to affect Ste6 trafficking. In contrast to wild-type Ste6, which was associated mainly with internal membranes, the ubiquitination-deficient mutants accumulated at the plasma membrane, as demonstrated by immunofluorescence and cell fractionation experiments. These findings suggest that ubiquitination is required for efficient endocytosis of Ste6 from the plasma membrane.

Transforming growth factor-alpha (TGF ?)-producing gastric carcinoma with acanthosis nigricans: An endocrine effect of TGF ? in the pathogenesis of cutaneous paraneoplastic syndrome and epithelial hyperplasia of the esophagus

A case of well-differentiated adenocarcinoma (Borrmann type 3) of the stomach in a 76-year-old man associated with the typical skin manifestations of acanthosis nigricans and with multiple protruding lesions showing epithelial hyperplasia of the esophagus is reported. The advanced tumor was located in the cardiac region of the stomach, and measured approximately 8 cm in diameter, with partial invasion to the esophagus. The associated cutaneous lesions were characterized by hyperpigmentation and by protruding verrucous papules on the torso, head, face, neck, upper extremities, perineum, and inguinal region. Histologically, the protruding skin lesions showed keratinocytes proliferation throughout the epidermis, resulting in diffuse hyperkeratosis, papillomatosis, and acanthosis of the skin. Immunohistological analysis showed coexpression of transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) receptors in the tumor from the stomach. It is reasonable to conclude from this evidence that gastric carcinoma cells secrete TGF alpha in an autocrine for auto-stimulation. EGF receptor expression was also noted on the papillomatous hyperplasia of the cutaneous lesion. Serum level of TGF alpha, determined by an enzyme-linked immunosorbent assay, was high (144 pg/ml; normal, 22.0 +/- 16 pg/ml (Mean +/- SD)). Serum TGF alpha abruptly decreased to 49 pg/ml on day 7 after the total gastrectomy, and then gradually increased to 77 pg/ml within 28 days. Amelioration of the cutaneous lesions and the protruding lesions in the esophagus was observed after surgical resection of the gastric carcinoma. This suggests that the TGF alpha stimulates the proliferation of keratinocytes involved with EGF receptor. Large amounts of circulating TGF alpha in the blood over a long period released by the primary tumor seem to act as an endocrine-like mechanism causing epidermal and esophageal epithelial cells to proliferate. There is a possible link in the pathogenesis of the acanthosis nigricans as a cutaneous paraneoplastic syndrome, and epithelial hyperplasia of the esophagus.

Expression of c-Myc, TGF-α and EGF-Receptor in Sporadic Medullary Thyroid Carcinoma

In 20 patients with sporadic medullary thyroid carcinoma (MTC), immuno-histochemistry was used to localize the expression of the c-Myc oncoprotein, transforming growth factor alpha (TGF-α) and epidermal growth factor receptor (EGFR) and these three markers were analysed regarding their relation to histopathological and histochemical variables of the tumours. The detection rates of c-Myc. TGF-α and EGFR were 90%, 90% and 95% respectively. Concomitant demonstration of the markers was reflected in significant associations (correlation factor between TGF-α and EGFR was 0.93, p < 0.001). The markers were almost invariably located within the cytoplasm, which might suggest their crucial role in growth regulation and cell differentiation; this seems especially true of TGF-α and EGFR. The different markers showed no relation to either histopathological or histochemical variables (tumour behaviour, tumour size, tumour cell type). The prominent co-expression of c-Myc, TGF-α and EGFR proteins indicates that in MTC these factors might be of importance for tumour cell proliferation via autocrine growth stimulation.

Phenotypic and functional characterization of long-lived NK cell lines of different maturational status obtained from mouse fetal liver.

Culture of day 14 mouse fetal liver (FL) cells in high dose IL-2, together with appropriate combinations of IL-4 and PMA, resulted in the generation of cell lines, termed FL-A lines, that were phenotypically and functionally indistinguishable from cultured adult splenic NK cell populations with the single important exception that no Ly49-expressing cells were present. By contrast, when FL cells were cultured in low-dose IL-2 alone, a second population of slow-growing NK-like cells, termed FL-B cells, emerged. These cells expressed the NK markers asialoGM1, 10A7, 2B4, and Fc gammaRII/III but differed from FL-A and splenic NK cells in expressing IL-2R alpha and stem cell factor receptor (SCFR) but no B220. Most lines derived in this manner had minimal or no cytolytic activity and only very low levels of NK1.1. However, they could secrete substantial quantities of several lymphokines including IL-3, granulocyte-macrophage (GM)-CSF, TNF-alpha, and, most surprisingly, IL-2. A minority of FL-B lines, typified by line 903, displayed marked cytolytic activity, moderate levels of NK1.1, reduced production of IL-2, and the capacity for accelerated growth in high-dose IL-2. FL-B lines generally expressed mRNA for CD3gamma but not for other CD3 chains, whereas FL-A and fetal thymic (FT) NK lines often expressed mRNA for all four CD3 chains. Despite many similarities to pro-T cells, FL-B cells showed no capacity to differentiate into mature T cells. Taken together, our results suggest that NK lines of different maturity can be obtained from fetal liver, with FL-B lines being the most immature, FL-A lines the most mature, and lines such as FL-B 903 representing an intermediate state of differentiation.

The sequence NPFXD defines a new class of endocytosis signal in Saccharomyces cerevisiae.

The yeast membrane protein Kex2p uses a tyrosine-containing motif within the cytoplasmic domain for localization to a late Golgi compartment. Because Golgi membrane proteins mislocalized to the plasma membrane in yeast can undergo endocytosis, we examined whether the Golgi localization sequence or other sequences in the Kex2p cytoplasmic domain mediate endocytosis. To assess endocytic function, the Kex2p cytoplasmic domain was fused to an endocytosis-defective form of the alpha-factor receptor. Ste2p. Like intact Ste2p, the chimeric protein, Stex22p, undergoes rapid endocytosis that is dependent on clathrin and End3p. Uptake of Stex22p does not require the Kex2p Golgi localization motif. Instead, the sequence NPFSD, located 37 amino acids from the COOH terminus, is essential for Stex22p endocytosis. Internalization was abolished when the N, P, or F residues were converted to alanine and severely impaired upon conversion of D to A. NPFSD restored uptake when added to the COOH terminus of an endocytosis-defective Ste2p chimera lacking lysine-based endocytosis signals present in wild-type Ste2p. An NPF sequence is present in the cytoplasmic domain of the a-factor receptor, Ste3p. Mutation of this sequence prevented pheromone-stimulated endocytosis of a truncated form of Ste3p. Our results identify NPFSD as a clathrin-dependent endocytosis signal that is distinct from the aromatic amino acid-containing Golgi localization motif and lysine-based, ubiquitin-dependent endocytosis signals in yeast.

In vitro activation of Stat3 by epidermal growth factor receptor kinase.

Stat proteins are SH2 domain-containing transcription factors that are activated in cells by various cytokines and growth factors. In the case of cytokines whose receptors lack protein kinase activity, phosphorylation-activation is mediated by members of the JAK family of tyrosine protein kinases. In the case of growth factors whose receptors have intrinsic tyrosine protein kinase activity, it is thought that Stat proteins can be activated either directly by the receptor or indirectly through JAK proteins. To test the possibility of direct activation, we have used purified Stat3 alpha, Stat3 beta, and epidermal growth factor receptor kinase produced in recombinant baculovirus-infected Sf9 insect cells. The Stat proteins formed a stable complex with the receptor kinase, and they were phosphorylated on tyrosine by the receptor kinase and activated for binding to DNA, properties shared with Stat proteins purified from Sf9 cells coexpressing JAK1 or JAK2. Both JAK-phosphorylated Stat3 beta and Stat3 beta phosphorylated in vitro by the receptor kinase were 20-50 times more active on a molar basis for DNA binding than phosphorylated Stat3 alpha. We conclude that Stat3 isoforms can be directly phosphorylated and thereby activated in vitro by the epidermal growth factor receptor kinase.

Transforming growth factor-alpha expression is altered during experimental hepatocarcinogenesis.

In order to characterize the role of transforming growth factor-alpha (TGF alpha) during hepatocarcinogenesis, liver tissue was examined at 10, 16, and 19 weeks following initial 10-week diethylnitrosamine (50 mg l-1 drinking water) exposure in female Wistar rats. Liver tissue protein extracts were electrophoresed and transferred to nitrocellulose filters. Levels of tissue-derived TGF alpha and epidermal growth factor receptor (EGFr) were assessed using an anti-TGF alpha monoclonal antibody (Ab-1) and an anti-EGFr polyclonal antibody (AB-4), coupled with scanning densitometric quantification. Immunolocalization of TGF alpha was performed in Bouin's-fixed, paraffin-embedded liver tissue sections. The distribution and intensity of TGF alpha immunoreactivity varied according to the degree of dysplasia, severely dysplastic cells being strongly immunoreactive. At week 10, mild hepatocyte dysplasia and perivenular inflammation were evident, together with a corresponding increase in perivenular TGF alpha immunoreactivity. By week 16, foci of moderate to severe dysplasia were observed; at this stage, there was a decrease in perivenular immunoreactivity but a further increase in overall liver tissue TGF alpha levels. Some 'altered foci' and dysplastic nodules showed intense immunoreactivity for TGF alpha. At these time points, immunodetectable liver EGFr was found to decrease significantly in comparison with normal control tissue. TGF alpha immunoreactivity was observed in fully developed carcinomas at week 19, although some tumours were negative by immunohistochemistry. The up-regulation of immunodetectable TGF alpha and the concomitant down-regulation of EGFr demonstrated positive (P < 0.01) and negative (P < 0.001) correlations, respectively, with hepatocyte proliferation indices. These findings suggest that the TGF alpha/EGFr ligand receptor system may be important during tumour promotion and in the stimulation of continued proliferation in hepatocellular carcinomas.

Mutation of Pro-258 in transmembrane domain 6 constitutively activates the G protein-coupled alpha-factor receptor.

The alpha-factor pheromone receptor stimulates MATa yeast cells to undergo conjugation. The receptor contains seven transmembrane domains that function in ligand binding and in transducing a signal to the cytoplasmic receptor sequences to mediate G protein activation. A genetic screen was used to isolate receptor mutations that constitutively signal in the absence of alpha-factor. The Pro-258-->Leu (P258L) mutation caused constitutive receptor signaling that was equivalent to about 45% of the maximum level observed in wild-type cells stimulated with alpha-factor. Mutations of both Pro-258 and the adjacent Ser-259 to Leu increased constitutive signaling to > or = 90% of the maximum level. Since Pro-258 occurs in the central portion of transmembrane domain 6, and since proline residues are expected to cause a kink in alpha-helical domains, the P258L mutation is predicted to alter the structure of transmembrane domain 6. The P258L mutation did not result in a global distortion of receptor structure because alpha-factor bound to the mutant receptors with high affinity and induced even higher levels of signaling. These results suggest that sequences surrounding Pro-258 may be involved in ligand activation of the receptor. Conformational changes in transmembrane domain 6 may effect a change in the adjacent sequences in the third intracellular loop that are thought to function in G protein activation. Greater than 90% of all G protein-coupled receptors contain a proline residue at a similar position in transmembrane domain 6, suggesting that this aspect of receptor activation may be conserved in other receptors.

Probing the functional conformation of the tridecapeptide mating pheromone of Saccharomyces cerevisiae through study of disulfide-constrained analogs.

Analogs of the Saccharomyces cerevisiae alpha-mating factor, Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr, where Lys7 and Gln10 were replaced with Cys, Cys(CH3), or Ser, were synthesized using solid-phase procedures on a phenylacetamidomethyl resin. Cyclo7,10[Cys7,X9,Cys10,Nle12]alpha-factor , where X=D-Val, D-Ala, L-Ala and Gly, were prepared by on-resin cyclization using thallic trifluoroacetate in yields of 20-30%. Linear sulfhydryl-containing peptides were generated from their corresponding cyclic peptide by treatment with dithioerythritol in basic solution. In the linear analogs, replacement of both Lys7 and Gln10 with a cysteine residue resulted in an over 100-fold loss of the biological activity when compared with the native pheromone. The corresponding cyclic disulfides were 5-10-fold more active than their sulfhydryl-containing homologs, and cyclo7,10[Cys7,L-Ala9,Cys10,Nle12] alpha-factor was 50-fold more potent than linear analogs containing Ser or Cys(CH3) in positions 7 and 10. Binding competition studies indicated that all analogs had low affinity for the alpha-factor receptor and there was a poor correlation between binding and activity in a growth arrest assay. A cyclic analog in which residues 8 and 9 were replaced by 5-aminopentanoic acid was not biologically active. Based on NMR studies, all cyclic peptides have a higher tendency to form beta-turns spanning residues 7-10 than their less active linear counterparts. The results provide strong evidence that this beta-turn is important for optimal signal transduction by alpha-factor.

Expression of growth factors and growth factor receptors in the liver of C57BL/10J mice following administration of phenobarbitone.

Liver enlargement is a common feature of non-genotoxic rodent hepatocarcinogens administered at high doses. In the present study, the expression of growth factors and growth factor receptors was investigated in the C57BL/1OJ mouse during liver enlargement induced by the non-genotoxic rodent hepatocarcinogen, sodium phenobarbitone (PB). Male mice were dosed 0-2500 p.p.m. PB in the diet for 1, 4 and 13 weeks. There was a dose and time dependent increase in liver weight. Hepatocyte replication, assessed by incorporation of bromodeoxyuridine, was increased in a dose-dependent manner at week 1 only (18-fold increase at 2000 p.p.m.) and was predominantly localized in the centrilobular region. At week 1, PB (2500 p.p.m.) caused transient increases in transforming growth factor alpha (TGFalpha) and epidermal growth factor receptor (EGFR) and decreases in transforming growth factor beta1 (TGF-beta1) and mannose-6-phosphate receptor (M6PR) in centrilobular hepatocytes which correlated with the replication in this region. At week 1, there was an increase in both hepatocyte growth factor (HGF) and hepatocyte growth factor receptor (HGFR) which colocalized in centrilobular hepatocytes; in some mice or periportal hepatocytes in other mice. After 13 weeks, HGF and HGFR were localized in the cytoplasm of centrilobular hepatocytes of all mice but exhibited a differential intracellular distribution across the lobule. At 2500 p.p.m. PB, EGFR and HGFR mRNA were essentially unchanged over the 13 week dosing period whilst M6PR mRNA was increased 2- to 4-fold. At 2500 p.p.m. PB, EGFR protein levels from immunoblots showed a consistent decrease over the 13 weeks whilst M6PR and HGFR protein levels were essentially unchanged. The protein level and mRNA data for EGFR suggest post-transcriptional modification. Thus, phenobarbitone caused transient replication of hepatocytes and modulation of growth stimulatory and inhibitory factors and their associated receptors in terms of overall levels and regional distribution in the liver.