CDC25: a component of the RAS-adenylate cyclase pathway in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae contains two functional homologues of the ras oncogene family, RAS1 and RAS2. These genes are required for growth, and all evidence indicates that this essential function is the activation of adenylate cyclase. In contrast, ras in mammalian cells does not appear to influence adenylate cyclase activity. To clarify the relation between ras function in yeast and in higher eukaryotes, and the role played by yeast RAS in growth control, it is necessary to identify functions acting upstream of RAS in the adenylate cyclase pathway. The evidence presented here indicates that CDC25, identified by conditional cell cycle arrest mutations, encodes such an upstream function.

Mammalian and yeast ras gene products: biological function in their heterologous systems.

Activated versions of ras genes have been found in various types of malignant tumors. The normal versions of these genes are found in organisms as diverse as mammals and yeasts. Yeast cells that lack their functional ras genes, RASSC-1 and RASSC-2, are ordinarily nonviable. They have now been shown to remain viable if they carry a mammalian rasH gene. In addition, yeast-mammalian hybrid genes and a deletion mutant yeast RASSC-1 gene were shown to induce morphologic transformation of mouse NIH 3T3 cells when the genes had a point mutation analogous to one that increases the transforming activity of mammalian ras genes. The results establish the functional relevance of the yeast system to the genetics and biochemistry of cellular transformation induced by mammalian ras genes.

Regulatory function of the Saccharomyces cerevisiae RAS C-terminus.

Activating mutations (valine 19 or leucine 68) were introduced into the Saccharomyces cerevisiae RAS1 and RAS2 genes. In addition, a deletion was introduced into the wild-type gene and into an activated RAS2 gene, removing the segment of the coding region for the unique C-terminal domain that lies between the N-terminal 174 residues and the penultimate 8-residue membrane attachment site. At low levels of expression, a dominant activated phenotype, characterized by low glycogen levels and poor sporulation efficiency, was observed for both full-length RAS1 and RAS2 variants having impaired GTP hydrolytic activity. Lethal CDC25 mutations were bypassed by the expression of mutant RAS1 or RAS2 proteins with activating amino acid substitutions, by expression of RAS2 proteins lacking the C-terminal domain, or by normal and oncogenic mammalian Harvey ras proteins. Biochemical measurements of adenylate cyclase in membrane preparations showed that the expression of RAS2 proteins lacking the C-terminal domain can restore adenylate cyclase activity to cdc25 membranes.

An adenylate cyclase from Saccharomyces cerevisiae that is stimulated by RAS proteins with effector mutations.

Conservative amino acid substitutions were introduced into the proposed effector regions of both mammalian Ha-ras (residues 32 to 40) and Saccharomyces cerevisiae RAS2 (residues 39 to 47) proteins. The RAS2[Ser 42] protein had reduced biological function in the yeast S. cerevisiae. A S. cerevisiae strain with a second-site suppressor mutation, SSR2-1, was isolated which could grow on nonfermentable carbon sources when the endogenous RAS2 protein was replaced by the RAS2[Ser 42] protein. The SSR2-1 mutation was mapped to the structural gene for adenylate cyclase (CYR1), and the gene containing SSR2-1 was cloned and sequenced. SSR2-1 corresponded to a point mutation that would create an amino acid substitution of a tyrosine residue for an aspartate residue at position 1547. The SSR2-1 gene encodes an adenylate cyclase that is dependent on ras proteins for activity, but is stimulated by Ha-ras and RAS2 mutant proteins that are unable to stimulate wild-type adenylate cyclase.

Genetic approaches to the determination of structure-function relationships of G protein-coupled receptors.

The beta-adrenergic receptor (beta AR), which has been extensively characterized pharmacologically, serves as a useful model system for the analysis of the structure-function relationships of G protein-coupled receptors. Genetic and biochemical analysis has revealed that the ligand binding domain of the receptor involves residues within the hydrophobic transmembrane core of the protein. Molecular substitution experiments suggest that adrenergic agonists and antagonists are anchored to the receptor through an ionic interaction between Asp113 in the third hydrophobic region of the receptor and the protonated amine group of the ligand. In addition, catecholamine agonists are bound through hydrogen bonding interactions between two serine residues in the fifth hydrophobic domain of the receptor and the catechol hydroxyl groups of the ligand. Agonist-mediated activation of the G protein Gs requires residues within the cytoplasmic loop linking the fifth and sixth transmembrane helices which are predicted to form amphipathic alpha-helices. The strong structural similarities among G protein-coupled receptors imply that the information gained from genetic analysis of the beta AR should be applicable to other hormone and neurotransmitter receptors of this class.

Design and synthesis of HIV protease inhibitors. Variations of the carboxy terminus of the HIV protease inhibitor L-682,679.

A series of tetrapeptide analogues of 1 (L-682,679), in which the carboxy terminus has been shortened and modified, was prepared and their inhibitory activity measured against the HIV protease in a peptide cleavage assay. Selected examples were tested as inhibitors of virus spread in cell culture. Compound 12 was a 10-fold more potent enzyme inhibitor than 1 in vitro and 30-fold more potent in inhibiting the viral spread in cells.

Thiol-beta-lactamase: replacement of the active-site serine of RTEM beta-lactamase by a cysteine residue.

We describe a procedure by which the codon (AGC) for the active-site serine-70 of pBR322 beta-lactamase (penicillinase, penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) is altered to that for cysteine (TGC). The pertinent nucleotide bases, A-G-C-A, positions 410-413, of pBR322 are excised by treating a limited HgiAI digest of pBR322 with the 3' leads to 5' exonuclease of T4 DNA polymerase. The new sequence, T-G-C-A, is inserted in two steps. First, the Kpn I molecular linker d(T-G-G-T-A-C-C-A) is ligated into the gap described above. The internal sequence G-T-A-C is then excised enzymatically with Kpn I and T4 DNA polymerase and the molecule is recircularized. This mutant gene, which codes for a thiol-beta-lactamase, confers on Escherichia coli K-12 hosts an ampicillin resistance that is reduced compared with that given by pBR322 yet is greater than that of E. coli lacking any intact beta-lactamase gene. Cell-free extracts of E. coli strains hosting the thiol-beta-lactamase gene possess a p-chloromercuribenzoate-sensitive beta-lactamase activity.

Structural basis of beta-adrenergic receptor function.

Receptors that mediate their actions by stimulating guanine nucleotide binding regulatory proteins (G proteins) share structural as well as functional similarities. The structural motif characteristic of receptors of this class includes seven hydrophobic putative transmembrane domains linked by hydrophilic loops. Genetic analysis of the beta-adrenergic receptor (beta AR) revealed that the ligand binding domain of this receptor, like that of rhodopsin, involves residues within the hydrophobic core of the protein. On the basis of these studies, a model for ligand binding to the receptor has been developed in which the amino group of an agonist or antagonist is anchored to the receptor through the carboxylate side chain of Asp113 in the third transmembrane helix. Other interactions between specific residues of the receptor and functional groups on the ligand have also been proposed. The interaction between the beta AR and the G protein Gs has been shown to involve an intracellular region that is postulated to form an amphiphilic alpha helix. This region of the beta AR is also critical for sequestration, which accompanies agonist-mediated desensitization, to occur. Structural similarities among G protein-linked receptors suggest that the information gained from the genetic analysis of the beta AR should help define functionally important regions of other receptors of this class.

Mapping the functional domains of the beta-adrenergic receptor.

The beta-adrenergic receptor (beta AR) serves as a model system for analysis of the structure-function relationships of G-protein-coupled receptors. Genetic analysis of the beta AR has demonstrated that the ligand-binding domain of this protein lies within the hydrophobic putative transmembrane core, involving specific amino acid residues in several of the transmembrane helices of the receptor. Site-directed mutagenesis of the receptor in conjunction with structural alterations of the ligands has revealed specific molecular interactions that are important for recognition of the ligand by the receptor. In addition, cytoplasmically exposed regions of the beta AR that are required for the activation of Gs have been identified. Because of the structural similarities among G-protein-coupled receptors, information gained from genetic analysis of the beta AR should prove useful in the development of specific agonists and antagonists for other receptors of this class.

Identification of effector residues and a neutralizing epitope of Ha-ras-encoded p21.

To identify the amino acid residues of the Harvey (Ha) ras-encoded protein that are involved in protein-protein interactions, we have created a series of mutant Ha-ras proteins. In particular, amino acid substitutions have been introduced within two regions, residues 32-42 and 61-80, that are conserved among ras proteins from different species. We observed that amino acid substitutions at positions 35, 36, 38, 40, and, to a lesser extent, 39 and 78 reduce the biological potency of Ha-ras protein in both mammalian and Saccharomyces cerevisiae cells, without noticeably affecting the known intrinsic biochemistry of these proteins. The reduction of in vivo activity for these mutant ras proteins correlates with their reduced ability to stimulate yeast adenylate cyclase. The ras-protein-neutralizing antibody Y13-259 binds to six residues: Glu-63, Ser-65, Ala-66, Met-67, Gln-70, and Arg-73. Single substitutions for these residues reduce Y13-259 antibody binding by at least a factor of 1000 but do not significantly affect biological activity. These data are discussed in terms of the model for Ha-ras protein based on the structure of the elongation factor EF-Tu-GDP complex.

Inhibition of yeast adenylate cyclase by antibodies to ras p21.

Monoclonal antibody Y13-259 to ras p21 was shown to bind to the highly conserved residues in the region 63-73 and to neutralize ras action in the Saccharomyces cerevisiae adenylate cyclase system. Inhibition of adenylate cyclase activity in isolated membranes by antibody Y13-259 occurred after a lag period of 6 min. This lag corresponded to the time necessary for binding of antibody Y13-259 to the membranes in a ras-dependent manner. The mechanism of inhibition appeared to be steric in nature because antibody Y13-259 neutralized ras p21 bound to a stable GTP analogue. Monoclonal antibodies Y13-4 and Y13-128 also inhibited yeast adenylate cyclase activity, and the epitopes for both the these antibodies were localized to ras region 65-75. However, the ras residues essential for binding of antibodies Y13-4 and Y13-128 to ras p21 (positions 65, 66, 68 and 75) were different from those essential for binding of antibody Y13-259 (positions 63, 65, 66, 67, 70 and 73). These results indicate that residues 63-75 constitute a major neutralizing epitope on ras p21.

Agonist-promoted sequestration of the beta 2-adrenergic receptor requires regions involved in functional coupling with Gs.

The molecular basis for the desensitization of beta 2-adrenergic receptors was investigated by oligonucleotide-directed mutagenesis. beta-Adrenergic receptor mutants containing deletions within the sixth hydrophilic domain that failed to couple to Gs and stimulate adenylyl cyclase did not undergo agonist-mediated sequestration. In contrast, all receptor mutants that displayed Gs coupling were sequestered away from the cell surface in response to isoproterenol. Progressive truncation of the C-terminus of the receptor resulted in decreases in the initial rates of receptor sequestration and functional uncoupling, although the final extent of these desensitization processes was not affected by the mutations. These data suggest that structural features of the beta 2-adrenergic receptor that are involved in receptor activation are also essential for mediating the subsequent inactivation caused by the sequestration of the receptor from the cell surface.

Intrinsic GTPase activity distinguishes normal and oncogenic ras p21 molecules.

The 21-kilodalton protein (p21) encoded by normal cellular Harvey-ras has been expressed in Escherichia coli as a fusion protein by using the pUC8 vector and has been purified to greater than 95% homogeneity by ion-exchange chromatography and gel filtration. The purified protein molecules possess intrinsic GTPase activity on the basis of the following criteria: (i) elution of the GTPase activity with p21 GDP-binding activity in two different chromatography systems, (ii) parallel thermal inactivation of GTPase activity and p21 GTP-binding activity, and (iii) immunoprecipitation of the GTPase activity with monoclonal antibodies to p21. At 37 degrees C, the rate of GTP hydrolysis by the purified normal p21 assayed in solution was 5.3-6.6 mmol/min per mol of p21. The rate of GTP hydrolysis by a form of p21 [Val12] encoded by a human oncogene was significantly lower (1.4-1.9 mmol/min per mol of p21). The presence of a threonine phosphate acceptor site at residue 59 also decreased p21 GTPase activity. For regulatory proteins that use GTP as part of their biochemical mechanism, the hydrolysis of GTP to GDP reverses the biological activity of the respective proteins. The observation that oncogenic forms of p21 lose GTPase activity suggests that GTP hydrolysis may be a biochemical event that inactivates the growth-promoting effects of a p21 X GTP complex.

Purification and properties of thiol beta-lactamase. A mutant of pBR322 beta-lactamase in which the active site serine has been replaced with cysteine.

The specifically mutated enzyme thiol beta-lactamase has been expressed in Escherichia coli by means of the trp promoter and purified to homogeneity. The gene for this enzyme results from a single base change N410 A----T in the gene of pBR322 RTEM beta-lactamase (EC 3.5.2.6, penicillinase, penicillin amido-beta-lactamhydrolase) which alters the codon for the active site Ser 70 to that for Cys. Precursor thiol beta-lactamase is processed to give the same NH2-terminal sequence as that for wild type enzyme. In contrast to the wild type enzyme, thiol beta-lactamase contains one free titratable thiol group/molecule. Thiol beta-lactamase catalyzes the hydrolysis of beta-lactams with a substrate specificity that is distinct from that of wild type enzyme. For benzyl-penicillin and ampicillin, the Km values are similar to wild type values although the kcat values are 1-2% that of wild type enzyme. For the cephalosporin nitrocefin, the Km is greater than 10-fold that of the wild type and the kcat is at least as large as the kcat for the wild type enzyme. Thiol beta-lactamase is different from wild type beta-lactamase in that it is not competitively inhibited by boric acid although a small degree of noncompetitive inhibition does occur. Whereas the circular dichroism spectra of both enzymes are nearly identical, thiol beta-lactamase at 40 degrees C is 3-fold more resistant to trypsin than is the wild type enzyme.

Yeast and mammalian ras proteins have conserved biochemical properties.

Mammalian ras oncogenes encode polypeptides of relative molecular mass (Mr) 21,000 (p21) which bind GTP and GDP. Oncogenic ras-encoded proteins differ from their normal homologues by an amino acid substitution for Gly 12, Ala 59 or Gln 61. Recently, we and others have observed that normal p21, encoded by the Ha-ras gene, has a GTP hydrolytic activity that is reduced by the oncogenic substitutions Val 12 or Thr 59. The yeast Saccharomyces cerevisiae contains two ras-related genes, RASsc1 and RASsc2, the expression of either of which is sufficient for viability. RASsc1 and RASsc2 encode proteins of 309 (SC1) and 322 (SC2) residues which are 62% homologous to mammalian p21 in their 172-amino acid N-terminal sequences. We report here that the N-terminal domain of SC1 binds GTP and GDP and has a GTP hydrolytic activity that is reduced in the variants SC1[Thr 66] and SC1[Leu 68] which are analogous to oncogenic Ha[Thr 59] and Ha[Leu 61], respectively. These results suggest that yeast and mammalian ras proteins have similar biochemical and possibly biological functions.

Identification of guanine nucleotides bound to ras-encoded proteins in growing yeast cells.

We have analyzed the guanine nucleotides bound to mammalian ras and yeast RAS proteins overexpressed in [32P]orthophosphate-labeled cultures of exponentially growing Saccharomyces cerevisiae cells. Whereas S. cerevisiae RAS1 and RAS2 proteins were immunoprecipitated bound entirely to GDP, mammalian Harvey ras was isolated with GTP and GDP bound in near-equimolar proportions. In a strain overexpressing a RAS2 variant where the RAS unique C-terminal domain was deleted, both GTP and GDP were detected in a ratio of 3:97. Increased amounts of GTP (16-75% of total guanine nucleotide) were observed bound to all ras proteins containing mutations that inhibit GTP hydrolytic activity. Increasing proportions of GTP bound to the various ras proteins correlated with increasing biological potency to bypass cdc25 lethality in yeast.

Mutations that uncouple the beta-adrenergic receptor from Gs and increase agonist affinity.

The deletion of residues 239-272 from the hamster beta-adrenergic receptor resulted in a loss of the ability of the receptor, expressed in mouse L cells, to stimulate adenylate cyclase (Dixon, R. A. F., Sigal, I. S., Rands, E., Register, R. B., Candelore, M. R., Blake, A. D., and Strader, C. D. (1987) Nature 326, 73-77). This mutant receptor (D(239-272)beta AR) bound the agonist isoproterenol with a single class of binding sites, in contrast to the wild-type beta-adrenergic receptor, which exhibited two classes of agonist affinity sites. We now report that the affinity of D(239-272)beta AR for isoproterenol is relatively insensitive to detergent solubilization or to treatment with either GTP or NaF, indicating the absence of a receptor-Gs interaction. Whereas deletions within the region of amino acids 229-258 did not reduce the ability of the receptor to couple to Gs or to stimulate adenylate cyclase, the deletion of either of the regions 222-229 or 258-270 resulted in receptors which were unable to couple to Gs. The affinities of D(222-229)beta AR, D(239-272)beta AR, and D(258-270)beta AR toward isoproterenol were greater than that observed for the low affinity, uncoupled form of the wild-type receptor. These results suggest a role for the regions of the beta-adrenergic receptor encompassing amino acids 222-229 and 258-270, which are predicted to form amphiphilic helices, in the agonist-promoted activation of Gs.

Identification of residues required for ligand binding to the beta-adrenergic receptor.

The functional significance of conserved polar amino acids within the putative transmembrane region of the beta-adrenergic receptor (beta AR) was examined by oligonucleotide-directed mutagenesis of the hamster gene encoding beta AR and expression of the mutant genes in COS-7 cells. Although a substitution of aspartate at position 113 with an asparagine residue did not affect expression or processing of the protein, the resulting mutant beta AR did not show detectable binding toward the antagonist iodocyanopindolol. Replacement of the aspartate and asparagine residues at positions 79 and 318, respectively, had no effect on the affinity of the receptor toward antagonists but reduced the affinity of the receptor toward agonists by 1 order of magnitude. Furthermore, we observed that substitution of the proline at position 323 with a serine residue resulted in improper or incomplete processing of the beta AR, presumably reflecting a role for this residue in the folding of the receptor. Together with our previous results from deletion mutagenesis studies, these observations indicate that the ligand binding site involves the transmembrane region of the beta AR.