Two different contact sites are recruited by cardiotrophin-like cytokine (CLC) to generate the CLC/CLF and CLC/sCNTFRalpha composite cytokines.
The cytokines of the interleukin-6 family are multifunctional proteins that regulate cell growth, differentiation, and other cell functions in a variety of biological systems including the immune, inflammatory, hematopoietic, and nervous systems. One member of this family, ciliary neurotrophic factor (CNTF), displays biological functions more restricted to the neuromuscular axis. We have recently identified two additional ligands for the CNTF receptor complex. Both are composite cytokines formed by cardiotrophin-like cytokine (CLC) associated to either the soluble type I cytokine receptor CLF or the soluble form of CNTF receptor alpha (CNTFRalpha). The present study was aimed at analyzing the interactions between the cytokine CLC and its different receptor chains. For this purpose, we modeled CLC/receptor interactions to define the residues potentially involved in the contact sites. We then performed site-directed mutagenesis on these residues and analyzed the biological interactions between mutants and receptor chains. Importantly, we found that CLC interacts with the soluble forms of CNTFRalpha and CLF via sites 1 and 3, respectively. For site 1, the most crucial residues involved in the interaction are Trp67, Arg170, and Asp174, which interact with CNTFRalpha. Surprisingly, the residues that are important for the interaction of CLC with CLF are part of the conserved FXXK motif of site 3 known to be the interaction site of LIFRbeta. Obtained results show that the Phe151 and Lys154 residues are effectively involved in the interaction of CLC with LIFRbeta. This study establishes the molecular details of the interaction of CLC with CLF, CNTFRalpha, and LIFRbeta and helps to define the precise role of each protein in this functional receptor complex.
Perret
David
D
INSERM U564, CHU d'Angers, 4 rue Larrey, 49033 Angers Cedex 01, France.
Guillet
Catherine
C
Elson
Greg
G
Froger
Josy
J
Plun-Favreau
Hélène
H
Rousseau
François
F
Chabbert
Marie
M
Gauchat
Jean-François
JF
Gascan
Hugues
H
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D017124:Conserved Sequence; D016207:Cytokines / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D015850:Interleukin-6 / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D020801:Receptor, Ciliary Neurotrophic Factor / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D016207:Cytokines; D015850:Interleukin-6; D020801:Receptor, Ciliary Neurotrophic Factor; D011994:Recombinant Proteins; C121169:cardiotrophin-like cytokine