Piebaldism is an autosomal dominant genetic disorder characterized by congenital patches of skin and har from which melanocytes are completely absent. A similar disorder of mouse, dominant white spotting (W), results from mutations of the c-Kit protooncogene, which encodes the receptor for mast/stem cell growth factor. We identified a KIT gene mutation in a proband with classic autosomal dint piebaldism. This mutation results in a Gly --Arg substitution at codon 664, within the tyrosine kinase domain. This substitution was not seen in any normal individuals and was completely linked to the piebald phenotype in the proband's family. Piebaldism in this family thus appears to be the human homologue to dominant white spotting (W) of the mouse.

The screening of combinatorial peptide libraries has emerged as an important tool in the discovery of novel substrates or ligands for enzyme and receptor targets. For example, screening linear peptide libraries using streptavidin as a model receptor system has previously identified many low-affinity peptide ligands, all of which contain the common motif His-Pro-Gln (HPQ). We reasoned that constraining the conformational freedom of linear peptides by cyclization in a library would yield peptide ligands of increased affinity. Three different cyclic peptide libraries were constructed in an M13 phage display system as N-terminal pIII protein fusions. The random peptide sequences were flanked by two cysteine residues, which allows efficient disulfide bond formation and cyclization during phage assembly. These cyclic peptide libraries were screened with streptavidin as the model receptor system. Many sequences, all of which contained the motif His-Pro-Gln (HPQ), were discovered, and in the preceding paper, the structures of complexes of streptavidin-bound cyclic and linear peptides are described (Katz, 1995). Analysis of binding kinetics and affinities demonstrated that the conformationally constrained cyclic peptides bound streptavidin with affinities up to 3 orders of magnitude higher than linear peptides identified in previous library screens. These results demonstrate the potential of screening conformationally constrained peptide libraries for high-affinity novel receptor ligands or enzyme substrates.

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We have probed the interaction of human erythropoietin (EPO) with its receptor (EPO-R) by analyzing a panel of 17 EPO mutants in a variety of in vitro assays. Mutant proteins were expressed in 293s cells and quantified by using an N-terminal epitope tag in conjunction with a surface plasmon resonance assay. Receptor binding was studied using both a soluble form of the EPO-R extracellular domain in an ELISA-format binding competition assay and full-length EPO-R in transfected BaF3 cells. Proliferative activity of the mutants was also determined in the BaF3-derived cell line and was correlated with the results from binding assays. Based on the results of these assays, we identified two distinct receptor binding sites on the EPO molecule. We propose that one site, containing residues Arg-150 and Lys-152, binds initially to EPO receptor on the cell surface. A second site, containing Arg-103 and , is involved in binding a second EPO-R at the cell surface, thus forming a homodimeric receptor complex. Furthermore, we demonstrate that one EPO mutant (R103A), which has previously been shown to lack proliferative function, is in fact an EPO antagonist. Taken together, these data support a sequential dimerization mechanism of EPO-R activation.Human erythropoietin (EPO) is a 166-aa glycoprotein that is involved in the proliferation and differentiation of erythroid progenitor cells (1). These cellular responses are mediated by the EPO receptor (EPO-R), a 508-aa glycoprotein containing a single transmembrane domain (2) that has been classified as a member of the growth hormone subfamily of class I cytokine receptors (3). Several studies have implicated receptor dimerization in the EPO signal transduction mechanism. For example, a constitutively active variant of EPO-R has been isolated, containing an arginine to cysteine mutation at position 129 in the receptor extracellular domain (4). The R129C mutant receptors have been shown to form disulfide-linked dimers in plasma membranes (5), and several similar cysteine mutations have been discovered that render the receptor constitutively active (6). EPO-R has also been coexpressed with inactive EPO-R analogs that lack most of the cytoplasmic domain, revealing a dominant inhibitory effect of the inactive receptor on proliferative activity of the wild-type (WT) receptor (6, 7). Recent biophysical studies (8) have also suggested that EPO binds to two receptor molecules, one with high affinity ("1 nM) and one with low affinity (-1 ,uM Although dimerization of EPO-R at the cell surface has not been directly observed, we sought to explore the EPO-R activation mechanism in terms of a sequential dimerization model, as has been described for human growth hormone (hGH) receptor. X-ray crystallography (9) and other biophysical techniques (10) have demonstrated that one hGH molecule binds to two receptor molecules, and mutagenesis studies (11-13) have revealed important receptor binding determinants on hGH. hGH binds initially to one receptor via residues comprising "site 1" on the ho...