Activation and inhibition of erythropoietin receptor function: role of receptor dimerization.
Members of the cytokine receptor superfamily have structurally similar extracellular ligand-binding domains yet diverse cytoplasmic regions lacking any obvious catalytic domains. Many of these receptors form ligand-induced oligomers which are likely to participate in transmembrane signaling. A constitutively active (factor-independent) mutant of the erythropoietin receptor (EPO-R), R129C in the exoplasmic domain, forms disulfide-linked homodimers, suggesting that the wild-type EPO-R is activated by ligand-induced homodimerization. Here, we have taken two approaches to probe the role EPO-R dimerization plays in signal transduction. First, on the basis of the crystal structure of the ligand-bound, homodimeric growth hormone receptor (GH-R) and sequence alignment between the GH-R and EPO-R, we identified residues of the EPO-R which may be involved in intersubunit contacts in an EPO-R homodimer. Residue 129 of the EPO-R corresponds to a residue localized to the GH-R dimer interface region. Alanine or cysteine substitutions were introduced at four other residues of the EPO-R predicted to be in the dimer interface region. Substitution of residue E-132 or E-133 with cysteine renders the EPO-R constitutively active. Like the arginine-to-cysteine mutation at position 129 in the exoplasmic domain (R129C), E132C and E133C form disulfide-linked homodimers, suggesting that constitutive activity is due to covalent dimerization. In the second approach, we have coexpressed the wild-type EPO-R with inactive mutants of the receptor missing all or part of the cytosolic domain. These truncated receptors have a dominant inhibitory effect on the proliferative action of the wild-type receptor. Taken together, these results strengthen the hypothesis that an initial step in EPO- and EPO-R-mediated signal transduction is ligand-induced receptor dimerization.
Watowich
S S
SS
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142.
Hilton
D J
DJ
Lodish
H F
HF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002999:Clone Cells; D003001:Cloning, Molecular; D003545:Cysteine; D018076:DNA, Complementary / Q000378:metabolism; D004921:Erythropoietin / Q000378:metabolism / Q000494:pharmacology*; D005091:Exons; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D007438:Introns; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D005810:Multigene Family; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D017467:Receptors, Erythropoietin / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D015398:Signal Transduction; D014162:Transfection
D018076:DNA, Complementary; D007377:Interleukin-3; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; D004921:Erythropoietin; D003545:Cysteine