The chemokine receptor CXCR4 is a co-receptor for T-tropic strains of HIV-1. A number of small molecule antagonists of CXCR4 are in development but all are likely to lead to adverse effects due to the physiological function of CXCR4. To prevent these complications, allosteric agonists may be therapeutically useful as adjuvant therapy in combination with small molecule antagonists. A synthetic cDNA library coding for 160,000 different SDF-based peptides was screened for CXCR4 agonist activity in a yeast strain expressing a functional receptor. Peptides that activated CXCR4 in an autocrine manner induced colony formation. Two peptides, designated RSVM and ASLW, were identified as novel agonists that are insensitive to the CXCR4 antagonist AMD3100. In chemotaxis assays using the acute lymphoblastic leukemia cell line CCRF-CEM, RSVM behaves as a partial agonist and ASLW as a superagonist. The superagonist activity of ASLW may be related to its inability to induce receptor internalization. In CCRF-CEM cells, the two peptides are also not inhibited by another CXCR4 antagonist, T140, or the neutralizing monoclonal antibodies 12G5 and 44717.111. These results suggest that alternative agonist-binding sites are present on CXCR4 that could be screened to develop molecules for therapeutic use.

Yeasts have three functionally redundant G 1 cyclins required for cell cycle progression through G 1 . Mutations in GIN4 and CLA4 were isolated in a screen for mutants that are inviable with deletions in the G 1 cyclins CLN1 and CLN2. cln1 cln2 cla4 and cln1 cln2 gin4 cells arrest with a cytokinesis defect; this defect was efficiently rescued by CLN1 or CLN2 expression. GIN4 encodes a protein with strong homology to the Snf1p serine/ threonine kinase. Cla4p is homologous to mammalian p21-activated kinases (PAKs) (kinases activated by the rho-class GTPase Rac or Cdc42). We developed a kinase assay for Cla4p. Cla4p kinase was activated in vivo by the GTP-bound form of Cdc42p. The specific activity of Cla4p was cell cycle regulated, peaking near mitosis. Deletion of the Cla4p pleckstrin domain diminished kinase activity nearly threefold and eliminated in vivo activity. Deletion of the Cla4p Cdc42-binding domain increased kinase activity nearly threefold, but the mutant only weakly rescued cla4 function in vivo. This suggests that kinase activity alone is not sufficient for full function in vivo. Deletion of the Cdc42-binding domain also altered the cell cycle regulation of kinase activity. Instead of peaking at mitosis, the mutant kinase activity exhibited reduced cell cycle regulation and peaked at the G 1 /S border. Cla4p kinase activity was not reduced by mutational inactivation of gin4, suggesting that Gin4p may be downstream or parallel to Cla4p in the regulation of cytokinesis.

The CLN1, CLN2 and CLN3 gene family of G1‐acting cyclin homologs of Saccharomyces cerevisiae is functionally redundant: any one of the three Cln proteins is sufficient for activation of Cdc28p protein kinase activity for cell cycle START. The START event leads to multiple processes (including DNA replication and bud emergence); how Cln/Cdc28 activity activates these processes remains unclear. CLN3 is substantially different in structure and regulation from CLN1 and CLN2, so its functional redundancy with CLN1 and CLN2 is also poorly understood. We have isolated mutations that alter this redundancy, making CLN3 insufficient for cell viability in the absence of CLN1 and CLN2 expression. Mutations causing phenotypes specific for the cell division cycle were analyzed in detail. Mutations in one gene result in complete failure of bud formation, leading to depolarized cell growth. This gene was identified as BUD2, previously described as a non‐essential gene required for proper bud site selection but not required for budding and viability. Bud2p is probably the GTPase‐activating protein for Rsr1p/Bud1p [Park, H., Chant, I. and Herskowitz, I. (1993) Nature, 365, 269‐274]; we find that Rsr1p is required for the bud2 lethal phenotype. Mutations in two other genes (ERC10 and ERC19) result in a different morphogenetic defect: failure of cytokinesis resulting in the formation of long multinucleate tubes. These results suggest direct regulation of diverse aspects of bud morphogenesis by Cln/Cdc28p activity.

In budding yeast, one of three G1 cyclins is required for progression though START, when cells commit to a further round of cell division. We have identified mutations in ALG1 (ERC14), a gene required for N-glycosylation, which are inviable in a cln1 cln2 background but are rescued by over-expression of CLNs. CLN1 and CLN2 are much more efficient than CLN3 in rescuing the erc14-1 allele. The erc14-1 allele results in a significant N-glycosylation defect, and no rescue of this defect by CLN1 over-expression was detected. These data suggest that CLN over-expression could be allowing cells to live with lower levels of N-glycosylation, possibly by overcoming a checkpoint sensitive to N-glycosylation capacity. A plasmid suppressor of alg1, PSA1, encodes a 361 amino-acid protein with homology to NDP-hexose pyrophosphorylases, the enzymes that catalyze the formation of activated sugar nucleotides. PSA1 is an essential gene, and PSA1 transcription is nearly co-ordinately regulated with CLN2 transcription, peaking near START. Co-ordinate regulation of glycosylation, sugar nucleotide metabolism, and cell-cycle progression through G1 may be a feature that ensures adequate cell-wall precursors are present before bud emergence.

A genetic screen in Saccharomyces cerevisiae identified mutations in mammalian adenylyl cyclase that activate the enzyme in the absence of G s ␣. Thirteen of these mutant proteins were characterized biochemically in an assay system that depends on a mixture of the two cytosolic domains (C 1 and C 2 ) of mammalian adenylyl cyclases. Three mutations, I1010M, K1014N, and P1015Q located in the ␤4-␤5 loop of the C 2 domain of type II adenylyl cyclase, increase enzymatic activity in the absence of activators. The K1014N mutation displays both increased maximal activity and apparent affinity for the C 1 domain of type V adenylyl cyclase in the absence of activators of the enzyme. The increased affinity of the mutant C 2 domain of adenylyl cyclase for the wild type C 1 domain was exploited to isolate a complex containing VC 1 , IIC 2 , and G s ␣-guanosine 5-3-O-(thio)triphosphate (GTP␥S) in the absence of forskolin and a complex of VC 1 , IIC 2 , forskolin, and P-site inhibitor in the absence of G s ␣-GTP␥S. The isolation of these complexes should facilitate solution of crystal structures of low activity states of adenylyl cyclase and thus determination of the mechanism of activation of the enzyme by forskolin and G s ␣.Mammalian adenylyl cyclases are membrane-bound enzymes that catalyze the synthesis of the intracellular second messenger cyclic AMP from ATP. Nine isoforms of the enzyme have been detected, and they display characteristic regulatory properties and patterns of cellular distribution (1, 2). Cellular rates of cyclic AMP synthesis are controlled by a variety of extracellular ligands that interact with heptahelical receptors in the plasma membrane. Relevant receptors can either stimulate cyclic AMP synthesis, usually via the intermediacy of a G protein 1 (G s ) that activates adenylyl cyclase, or inhibit cyclic AMP synthesis, often by interaction with an inhibitory G protein, G i . Mammalian adenylyl cyclases can also be activated by the diterpene forskolin (3) and inhibited by certain adenosine analogs and adenine nucleotides called P-site inhibitors (4). Certain adenylyl cyclases are also regulated by Ca 2ϩ , Ca 2ϩ -calmodulin, and phosphorylation (5).Mammalian adenylyl cyclases are integral membrane proteins that appear to contain two sets of six membrane-spanning helices that are separated by a large (ϳ40 kDa) cytoplasmic loop and followed by a similarly sized carboxyl-terminal cytosolic domain (6). The cytosolic domains, termed C 1 and C 2 , have been extensively studied; they are responsible for catalytic activity and most of the regulatory properties of the enzymes (7). The first 200 -250 amino acids of each cytosolic domain, designated C 1a and C 2a , are the most highly conserved regions among adenylyl cyclases. Strikingly, the C 1a and C 2a domains are approximately 50% similar and 25% identical to each other within a single isoform of adenylyl cyclase, and they are 20 -25% similar to the catalytic domains of membrane-bound and cytosolic guanylyl cyclases.The C 1a and C 2a domains of adenylyl cyclas...