Characterization of the Receptor Binding Sites of Human Leukemia Inhibitory Factor and Creation of Antagonists

doi:10.1074/jbc.271.20.11971

Characterization of the Receptor Binding Sites of Human Leukemia Inhibitory Factor and Creation of Antagonists (*)

From the (1)CRC Growth Factor Group, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom and the
(2)Department of Biochemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom

^¶ To whom correspondence should be addressed. Tel.: 44(0)-1214-147533; Fax: 44(0)-1214-143982; j.k.heath{at}bham.ac.uk.

Abstract

Residues in human leukemia inhibitory factor (hLIF) crucial for binding to both the human LIF receptor (R) and gp130 were identified by analysis of alanine scanning mutants of hLIF in assays for both receptor binding and bioactivity. The region of hLIF most important for binding to the hLIF-R is composed of residues from the amino terminus of the D-helix, carboxyl terminus of the B-helix, and C-D loop. This site forms a distinct surface at the end of the four-helix bundle in the tertiary structure of the closely related murine LIF. The two residues of hLIF that contribute the majority of free energy for hLIF-R binding, Phe-156 and Lys-159 are surrounded by other residues which have only a moderate impact. This arrangement of a few key residues surrounded by less important ones is analogous to the functional binding epitope of human growth hormone for its receptor. A second region of hLIF that includes residues from the carboxyl terminus of the D-helix and A-B loop also had a weak influence on hLIF-R binding. Residues in hLIF from both the A- and C-helices are involved in binding the gp130 co-receptor. Abolition of the gp130 binding site in hLIF created antagonists of LIF action.

Footnotes

↵^§ Funded by a fellowship from the American Cancer Society.
↵* This research was supported in part by the Cancer Research Campaign. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

LIF

leukemia inhibitory factor

m

murine

h

human

LIF-R

LIF receptor

CNTF

ciliary neurotrophic factor

CT

cardiotrophin

IL

interleukin

CSF

colony-stimulating factor

hGH

human growth hormone

GHR

growth hormone receptor

PCR

polymerase chain reaction

PAGE

polyacrylamide gel electrophoresis

PBS

phosphate-buffered saline

BSA

bovine serum albumin

CPK

Corey-Pauling-Koltun

Fc

constant region domain of human -immunoglobulin

IgG

-immunoglobulin.
↵²R. C. Robinson et al., submitted for publication.
↵³A. B. Vernallis, K. R. Hudson, and J. K. Heath, manuscript in preparation.
- Received November 8, 1995.
- Revision received January 22, 1996.