Secreted peptide ligands are known to play key roles in the regulation of plant growth, development, and environmental responses. However, phenotypes for surprisingly few such genes have been identified via loss-of-function mutant screens. To begin to understand the processes regulated by the CLAVATA3 (CLV3)/ESR (CLE) ligand gene family, we took a systems approach to gene identification and gain-of-function phenotype screens in transgenic plants. We identified four new CLE family members in the Arabidopsis (Arabidopsis thaliana) genome sequence and determined their relative transcript levels in various organs. Overexpression of CLV3 and the 17 CLE genes we tested resulted in premature mortality and/or developmental timing delays in transgenic Arabidopsis plants. Overexpression of 10 CLE genes and the CLV3 positive control resulted in arrest of growth from the shoot apical meristem (SAM). Overexpression of nearly all the CLE genes and CLV3 resulted in either inhibition or stimulation of root growth. CLE4 expression reversed the SAM proliferation phenotype of a clv3 mutant to one of SAM arrest. Dwarf plants resulted from overexpression of five CLE genes. Overexpression of new family members CLE42 and CLE44 resulted in distinctive shrub-like dwarf plants lacking apical dominance. Our results indicate the capacity for functional redundancy of many of the CLE ligands. Additionally, overexpression phenotypes of various CLE family members suggest roles in organ size regulation, apical dominance, and root growth. Similarities among overexpression phenotypes of many CLE genes correlate with similarities in their CLE domain sequences, suggesting that the CLE domain is responsible for interaction with cognate receptors.

Residues in human leukemia inhibitory factor (hLIF) crucial for binding to both the human LIF receptor (R) and gp130 were identified by analysis of alanine scanning mutants of hLIF in assays for both receptor binding and bioactivity. The region of hLIF most important for binding to the hLIF-R is composed of residues from the amino terminus of the D-helix, carboxyl terminus of the B-helix, and C-D loop. This site forms a distinct surface at the end of the four-helix bundle in the tertiary structure of the closely related murine LIF. The two residues of hLIF that contribute the majority of free energy for hLIF-R binding, Phe-156 and Lys-159 are surrounded by other residues which have only a moderate impact. This arrangement of a few key residues surrounded by less important ones is analogous to the functional binding epitope of human growth hormone for its receptor. A second region of hLIF that includes residues from the carboxyl terminus of the D-helix and A-B loop also had a weak influence on hLIF-R binding. Residues in hLIF from both the A-and C-helices are involved in binding the gp130 co-receptor. Abolition of the gp130 binding site in hLIF created antagonists of LIF action.Leukemia inhibitory factor (1-3) is a secreted cytokine that elicits pleiotropic effects on a diverse range of cell types, these include embryonic stem cells, primordial germ cells, neurons, adipocytes, hepatocytes, and osteoblasts (4, 5). In mice, gene knockout experiments have demonstrated that LIF 1 is essential for embryonic implantation (6, 7). In contrast to the mild phenotype in mice lacking the LIF gene, the targeted deficiency of the specific LIF receptor (LIF-R), results in mice that have multiple placental, skeletal, neural, and metabolic disorders, which cause perinatal death (8). The biological differences between these two genetic deficiencies is an outcome of the use of the LIF-R for signal transduction by several ligands. The cytokines known to bind to the LIF-R are included in a group that share some biological properties with LIF: oncostatin M, ciliary neurotrophic factor (CNTF), cardiotrophin (CT-1), interleukin-6 (IL-6), and interleukin-11 (9 -14).The crystal structure of recombinant murine LIF (mLIF) has been solved (15). LIF has a four ␣-helical bundle topology with up-up-down-down helix orientation that has long crossover loops between the first two and last two helices. The structure of LIF shows greatest homology to granulocyte-colony-stimulating factor (G-CSF; Ref. 16), human growth hormone (hGH; Ref. 17) and the recently determined CNTF (18). These proteins all belong to the hematopoietin cytokine family, which is characterized by the four-helix bundle structure, but limited sequence homology between family members (19 -21). The hematopoietin cytokine family is divided into the short and long chain subfamilies (21). The long chain group of which both LIF and hGH are members is characterized by helices of approximately 25 residues, the presence of short helical regions in the long loops, and the complete absence o...

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The superantigen staphylococcal enterotoxin A (SEA) binds to major histocompatibility complex (MHC) class II molecules at two sites on either side of the peptide groove. Two separate but cooperative interactions to the human class II molecule HLA-DR1 were detected. The first high affinity interaction to the DR1 beta chain is mediated by a zinc atom coordinated by H187, H225, and D227 in SEA and H81 in the polymorphic DR1 beta chain. The second low affinity site is to the DR1 alpha chain analogous to SEB binding and is mediated by residue F47 in SEA. Binding of one SEA to the DR1 beta chain enhances the binding of a second SEA molecule to the DR1 alpha chain. The zinc site is on the opposite side of the SEA molecule from residue F47 so that one SEA molecule can readily bind two class II molecules. Both binding sites on SEA are required for maximal activity. Thus, unlike, SEB, SEA requires two separate binding sites for optimal activity, which may allow it to stabilize SEA interaction with T cell receptors, as well as to activate the antigen-presenting cell by cross-linking MHC class II.