Expression, crystallization and derivatization of the complete extracellular domain of the beta(c) subunit of the human IL-5, IL-3 and GM-CSF receptors.
The major signalling entity of the receptors for the haemopoietic cytokines granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and interleukin-5 (IL-5) is the shared beta(c) receptor, which is activated by ligand-specific alpha receptors. The beta(c) subunit is a stable homodimer whose extracellular region consists of four fibronectin domains and appears to be a duplication of the cytokine receptor homology module. No four domain structure has been determined for this receptor family and the structure of the beta(c) subunit remains unknown. We have expressed the extracellular domain in insect cells using the baculovirus system, purified it to homogeneity and determined its N-terminal sequence. N-glycosylation at two sites was demonstrated. Crystals of the complete domain have been obtained that are suitable for X-ray crystallographic studies, following mutagenesis to remove one of the N-glycosylation sites. The rhombohedral crystals of space group R3, with unit cell dimensions 186.1 A and 103.5 A, diffracted to a resolution of 2.9 A using synchrotron radiation. Mutagenesis was also used to engineer cysteine substitution mutants which formed isomorphous Hg derivatives in order to solve the crystallographic phase problem. The crystal structure will help to elucidate how the beta(c) receptor is activated by heterodimerization with the respective alpha/ligand complexes.
Gustin
S E
SE
Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Canberra, ACT, Australia.
Church
A P
AP
Ford
S C
SC
Mann
D A
DA
Carr
P D
PD
Ollis
D L
DL
Young
I G
IG
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons; D006031:Glycosylation; D006801:Humans; D007525:Isoelectric Focusing; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis / Q000737:chemistry*; D018123:Receptors, Interleukin / Q000096:biosynthesis / Q000737:chemistry*; D016185:Receptors, Interleukin-3 / Q000096:biosynthesis / Q000737:chemistry*; D053649:Receptors, Interleukin-5; D011994:Recombinant Proteins / Q000737:chemistry; D020539:Sequence Analysis, Protein; D013997:Time Factors
D018076:DNA, Complementary; D008024:Ligands; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D018123:Receptors, Interleukin; D016185:Receptors, Interleukin-3; D053649:Receptors, Interleukin-5; D011994:Recombinant Proteins; D003545:Cysteine