Saturation mutagenesis of the WSXWS motif of the erythropoietin receptor.
The WSXWS motif in the extracellular domain defines members of the cytokine receptor family, yet its role in receptor structure and function remains unresolved. To address this question we have generated a panel of 100 mutants within the WSXWS motif of the erythropoietin receptor, which represents all single amino acid substitutions of these five amino acids. All mutants were synthesized at the same level; however, their passage from the endoplasmic reticulum to the Golgi apparatus differed. Because of this, expression of mutant receptors at the cell surface varied more than 300-fold. The tolerance of the tryptophan and serine residues to substitution was quite narrow; as a result, most of these mutants were retained in the endoplasmic reticulum and showed no cell surface expression or reduced cell surface expression. Although many mutants with substitutions at the middle residue of the motif reached the cell surface, it was notable that one mutant, A234E, was processed more efficiently than the wild type receptor and was expressed in elevated numbers at the cell surface. Despite this variation, all mutant receptors that reached the cell surface appeared able to bind erythropoietin and transduce a proliferative signal normally. These results are discussed in terms of a general model for WSXWS function in which the motif contributes to efficient receptor folding.
Hilton
D J
DJ
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA.
Watowich
S S
SS
Katz
L
L
Lodish
H F
HF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004921:Erythropoietin / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017467:Receptors, Erythropoietin / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019699:Thrombospondins; D014162:Transfection
D008562:Membrane Glycoproteins; D009838:Oligodeoxyribonucleotides; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; D019699:Thrombospondins; D004921:Erythropoietin