Osamu Hiraoka

Requirement for the Immunoglobulin-like Domain of Granulocyte Colony-stimulating Factor Receptor in Formation of a 2:1 Receptor-Ligand Complex

The extracellular portion of the granulocyte colony-stimulating factor (G-CSF) receptor has a mosaic structure of six domains (each approximately 100 amino acid residues) consisting of an immunoglobulin-like (Ig) domain, a cytokine receptor homologous region subdivided into amino-terminal (BN) and carboxyl-terminal (BC) domains, and three fibronectin type III repeats. In the present study, we expressed the Ig-BN and the BN-BC regions and purified them to homogeneity as monomers using G-CSF affinity column chromatography. Using gel filtration high performance liquid chromatography, we investigated the molecular composition of receptor-ligand complexes formed between G-CSF and purified BN-BC or Ig-BN domains. In contrast to the well characterized example of the human growth hormone (GH) receptor, in which the BN-BC•GH complex shows a 2:1 receptor-ligand complex stoichiometry, the BN-BC domain of the G-CSF receptor formed a 1:1 complex. The isolated Ig-BN domain also formed a 1:1 complex with G-CSF. However, in the presence of both Ig-BN and BN-BC domains, we detected a 1:1:1 Ig-BN•G-CSF•BN-BC complex corresponding to the 2:1 receptor:ligand stoichiometry. These results suggest that 1) the Ig domain and both the BN and the BC domains are required for oligomerization of the G-CSF receptor, 2) G-CSF contains two binding sites for its receptor, and 3) there are two ligand binding sites on the G-CSF receptor, one site on the BN-BC domain and one on the Ig-BN domain.

Molecular cloning and expression of the cDNA coding for a new member of the S100 protein family from porcine cardiac muscle

We isolated a new calcium‐binding protein from porcine cardiac muscle by calcium‐dependent hydrophobic and dye‐affinity chromatography. It showed an apparent molecular weight of 11 000 on SDS‐PAGE. Amino acid sequence determination revealed that the protein contained two calcium‐binding domains of the EF‐hand motif. The cDNA gene coding for this protein was cloned from the porcine lung cDNA library. Sequence analysis of the cloned cDNA showed that the protein was composed of 99 amino acid residues and its molecular weight was estimated to be 11 179. Immunological and functional characterization showed that the recombinant S100C protein expressed in Escherichia coli was identical to the natural protein. Homologies to calpactin light chain, S100α and β protein were 41.1%, 40.9% and 37.5%, respectively. The protein was expressed at high levels in lung and kidney, and low levels in liver and brain. The tissue distribution was apparently different from those of the other S100 protein family. These results indicate that this protein represents a new member of the S100 protein family, and thus we refer to it as S100C protein.

EVIDENCE FOR THE LIGAND-INDUCED CONVERSION FROM A DIMER TO A TETRAMER OF THE GRANULOCYTE COLONY-STIMULATING FACTOR RECEPTOR

An extracellular portion of granulocyte colony‐stimulating factor (G‐CSF) receptor, which contains an immunoglobulin‐like (Ig) domain and cytokine receptor homologous (CRH) region, was secreted into the medium using Trichoplusia ni‐Autographa californica nuclear polyhedrosis virus system. The gene product was purified to homogeneity mainly as a dimer (85 kDa) using G‐CSF affinity column chromatography and gel filtration HPLC, although the product existed as a monomer (45 kDa) in the medium. Scatchard analyses suggested that only the dimer had high affinity ligand binding (K d = about 100 pM), which is comparable with the K d value of the cell surface receptor. The binding of G‐CSF to Ig‐CRH induced its tetramerization (200–250 kDa). The molecular composition of the tetrameric complex showed a stoichiometry of four ligands bound to four Ig‐CRH. These results suggested that the oligomeric mechanism of the G‐CSF receptor differs from that reported for growth hormone (GH) receptor, although CD spectrum spectroscopy suggested that the Ig‐CRH has a GH receptor‐like structure.

Biotin derivatives of endothelin: utilization for affinity purification of endothelin receptor.

Three different types of biotinylated endothelin 1 (ET-1) derivatives, [Cys1]-biotinylated ET-1, [Lys9]-biotinylated ET-1, and [Cys1][Lys9]-dibiotinylated ET-1, were obtained when the biotinylation reaction was carried out with sulfosuccinimidyl-6-(biotinamido)hexanoate in an aqueous solvent. The binding of [Lys9]-biotinylated ET-1 to the ET receptor was as efficient as that of natural ET-1, whereas the binding of either [Cys1]-biotinylated ET-1 or [Cys1][Lys9]-dibiotinylated ET-1 was significantly reduced. When ET-1 was reacted with succinimidyl-6-(biotinamido)hexanoate in an organic solvent, ET-1 was exclusively modified at lysine 9. The ET receptor was then isolated from human placenta by affinity chromatography with [Lys9]-biotinylated ET-1 and avidin-agarose. The purified ET receptor was active in ET binding and was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into two polypeptides with apparent molecular masses of 45 and 35 kDa. The NH2-terminal amino acid sequence indicated that the two polypeptides were from an identical subtype of the ET receptor (ETB, the ligand-nonselective type). A signal peptide from Met1 to Gly26 was missing from the 45-kDa ETB, whereas 64 amino acids at the NH2 terminus were missing from the 35-kDa ETB due to proteolytic cleavage which occurred between Arg64 and Ser65. Indeed, incubation of purified ETB with endopeptidase Arg-C resulted in degradation of the 45-kDa ETB, giving rise to the 35-kDa species by a specific cleavage at Arg64. The 35-kDa ETB was active in binding to ET-1, indicating that the NH2-terminal 64-amino-acid residues are not essential for ligand binding.(ABSTRACT TRUNCATED AT 250 WORDS)