Purification of a ligand for the EPH-like receptor HEK using a biosensor-based affinity detection approach.

Abstract

The pivotal role of receptor tyrosine kinases (RTKs) in regulation of cellular growth and differentiation has triggered considerable interest in the identification of novel members of this ubiquitous protein family. Screening techniques, which were not dependent on function, brought about the isolation of numerous novel RTKs for which the ligands were not known. Many were EPH-like RTKs, which comprise the largest RTK family known to date (1-19).In contrast to the PCR-based approaches used for most other EPH-like RTKs, human EPH-like kinase (HEK) was identified on the cell surface of a human pre-B-cell line with monoclonal antibody (mAb) IIIA4 (2). HEK protein was affinity purified on a mAb IIIA4 column (2), and its amino acid sequence revealed homology with EPH and the predicted amino acid sequences of other EPH-like RTKs [ELK (3), ECK (4), and ERK (5)]. The sequences of the mouse (MEK4) and chicken (CEK4) homologues of HEK have also been reported (6, 7). To date, at least 28 members of the EPH subfamily have been identified in diverse vertebrate species including zeThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Dissecting the EphA3/Ephrin-A5 Interactions Using a Novel Functional Mutagenesis Screen

“…Analyzing secreted mutant proteins during the primary colony screen, both functionally (for compromised ephrin-A5 binding) and immunologically (for native conformation), allowed an immediate and efficient selection of receptor mutants (7%) with altered ligand interaction sites and exclusion of those with a more global disruption of their overall domain structure. Effective isolation of mutants with intact global protein architecture was facilitated largely by selecting mutant proteins through their capacity of binding IIIA4, a mAb previously used successfully to monitor the biological integrity of recombinant EphA3 (21). Sequencing of 84% (38/45) of the clones recovered from this screen revealed, either by alignment to known EphB2/ephrin-B2 interaction surfaces or by functional assay, that in 37/38 cases at least one of the mutated amino acids is either directly involved with or falls within the immediate vicinity of predicted Eph/ephrin contacts (Table III).…”

“…The analysis of 677 informative colonies suggests that 50% of the clones produced proteins with apparent defects in ephrin-A5 binding (Table II). To evaluate whether any mutations disrupt the overall protein fold, we used the IIIA4 anti-EphA3 mAb that has been used previously to monitor the EphA3 conformation (21). This mAb binds only to the properly folded N-terminal globular domain of the receptor (11).…”

“…All proteins used for kinetic analysis were purified by IIIA4 affinity chromatography (21). The proteins were analyzed by SDS-PAGE/silver staining and Western blot using ␣-EphA3 (polyclonal) or ␣-FLAG (monoclonal) antibodies.…”

“…The protein concentrations were determined at A 215 (21). To ascertain the performance of sensor chip-immobilized proteins, samples of purified EphA3 ECD (62.5-1000 ng/ml) were analyzed in each assay.…”

“…Yeast EphA3 Expression Construct-A cDNA encoding the EphA3 extracellular domain (Glu 21 to Ser 539 , ECD) was cloned into the yeast expression vector YEpFLAG-1 (Sigma), containing a FLAG epitope and the alcohol dehydrogenase promoter for activation of transcription/ translation under low glucose conditions. In addition to a C-terminal myc epitope (EEQKLISEEDL*), EcoRI and BglII cloning sites were introduced between the FLAG and Myc sequences to accommodate insertion of EphA3-ECD cDNAs.…”