Worm Breeder's Gazette 12(4): 25 (October 1, 1992)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

A Structure/Function Study of the let-23 EGF Receptor Gene

Raffi V. Aroian[1], Giovanni M. Lesa[2], Paul W. Sternberg[2]

Figure 1

Figure 2

[1]Howard Hughes Medical Inst. and Division of Biology, CalTech, Pasadena, CA 91125
present address: Dept. of Biochemistry and Biophysics, U. of California, San Francisco, CA 94143-0448

[2]Howard Hughes Medical Inst. and Division of Biology, CalTech, Pasadena, CA 91125

The EGF receptor tyrosine kinase family, including the C. elegans let-23 protein, is characterized by several regions of similarity: (1) in the N-terminus, a ligand binding domain sandwiched between two cysteine-rich domains; (2) a single transmembrane (tm) domain; and (3) a kinase domain near the C-terminus. [See Figure 1]

The spacing between cysteines in the cysteine-rich domains is highly conserved among family members, and there is limited amino acid identity in the ligand binding domain. In addition to containing the residues shared among all tyrosine kinases, there are amino acid identities in the kinase domain unique to the family. The very C-terminus of the EGF receptor family, although not well conserved, is thought to contain elements important for regulation of kinase activity, receptor degradation, and substrate specificity.

We have sequenced 13 genetically characterized EMS alleles of let-23 to learn more about how EGF receptor sequence relates to function. Eight of these alleles are nulls, two are hypomorphs, one is defective in negative regulation, and two show tissue-specific defects ( let-23 functions in at least five tissues) that cannot be explained by a decrease in let-23 dosage alone.

We localized the molecular defect associated with these alleles (except sy1 )using hydroxylamine mismatch detection (protocol from R. Barstead). sy1 escaped mismatch detection and was sequenced directly. For all alleles, sequencing was performed using PCR-amplified DNA isolated from both homozygous and heterozygous stocks. For each allele we have detected only one mutation. The results are shown below. [See Figure 2]

Our results suggest that: (1) the conserved cysteines in the N-terminus are individually important for function ( sy10 / sy12 , mn23 , sy11 ); (2) one of the few conserved residues in the ligand binding domain is important for function ( mn216 );(3) let-23 has kinase activity since the alteration of amino acids conserved among kinases eliminates function ( sy16 , sy7 );and (4) the very C-terminus is important for tissue-specificity ( sy97 , sy1 ).This study demonstrates that C. elegans can provide valuable and novel insights into the relationship between primary sequence and function for the EGF receptor family.

Figure 1

Figure 2