Abstract
Ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) are cytokines that give rise to an identical set of tyrosine-phosphorylated proteins upon addition to responsive cells. One of these proteins is the interleukin-6 signal-transducing molecule gp130, which is required for signal transduction by both CNTF and LIF. Here we identify another prominent tyrosine-phosphorylated protein as LIF receptor (LIFR) beta, which was originally cloned as a LIF-binding protein. Cross-linking experiments with iodinated factors were carried out on a cell line responsive to CNTF and LIF, as well as on COS cells that were cotransfected with various combinations of gp130, LIFR beta, and CNTF receptor (CNTFR) alpha, the previously cloned CNTF-binding protein. These experiments reveal that LIF cross-links to LIFR beta alone, as well as to gp130 when it is coexpressed with LIFR beta. However, cross-linking of CNTF to LIFR beta and gp130 is only observed in the presence of CNTFR alpha. These and other data show that the two known LIF receptor components are recruited by CNTF and CNTFR alpha to form a trimeric CNTF receptor complex.
Highlights
Ponents ( 7 ) .CLIP2 has beenidentified as gp130 using monoclonal antibodies, anwde have suggestedthat CLIPlis LIFRP based on its molecular weight and the identity of the phosphorylation patterninduced by leukemia inhibitory factor (LIF) and Ciliary neurotrophic factor (CNTF)
We proposed that a functional CNTF receptor is composed of CNTFRa, gp130, and LIFRp( 7 ) .Since functional LIFreceptors appear to requireonly gp130 and LIFRP [8,9], a consequence of this model is that a functional LIF receptor canbe converted into a functional CNTF receptor by the presence of CNTFRa (4, 7, 10, 11). in a sense, the LIF signaland LIF, as well as on COS cells that were cotrans- transducing machinery,which occurs with widespread distrifected with various combinationsof gp130,LIFRB, and bution, would be appropriated for use by CNTF in the re
CNTF receptor (CNTFR) a, the previously cloned stricted setof cells that express CNTFRa[11].Recently, CNTF-bindingprotein. These experiments reveal that we have shown that CNTFRa can functionwhen present in LIF cross-links to LIFRB alone, as well as to gp130 asoluble form[10]
Summary
Superdex HR-75 column (PharmaciaLKB Biotechnology, Inc.) to allow separation of monomer from dimer; the monomeric fraction was used in all experiments. IF c PEP nonradioactive competitor in a 10-min incubation. Gation for 15 min at top speed in a microcentrifuge, the lysate was either subjected to SDS-PAGE directly or incubated overnight at 4 "C in the indicated antibody, 1 hin goat anti-mouse IgG conjugated to agarose (for a-gpl; Sigma) or protein A-Sepharose (for a-LIFRS; Pharmacia).The beads were washed 3 timesin 1ml of lysis buffer, boiled in sample buffer and subjected to SDSpolyacrylamide gel electrophoresis on gels containing 7% acrylamide. B , EW-1 cells were incubated with the indicated factor and cross-linked as described under "Experimental Procedures," immunoprecipitated with the indicated antibody, subjected to SDS-PAGE, analyzed by autoradiography. L or C denote the addition of nonradioactive LIF or CNTF, respectively,as a competitor. Cross-linked products corresponding to LIFRP (*), gp130 (m), or CNTFRa (+) are indicated.
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