SPECIFICITY OF LIM DOMAIN INTERACTIONS WITH RECEPTOR TYROSINE KINASES

LIM domains, Cys-rich motifs containing approximately 50 amino acids f ound in a variety of proteins, are proposed to direct protein protein interactions, To identify structural targets recognized by LIM domains , we have utilized random peptide library selection, the yeast two-hyb rid system, and glutathione S-transferase fusions, Enigma contains thr ee LIM domains within its carboxyl terminus and LIM3 of Enigma specifi cally recognizes active but not mutant endocytic codes of the insulin receptor (InsR) (Wu, R Y,, and Gill, Gr, N, (1994) J. Biol, Chem. 269, 2508-25090). Interaction of two random peptide libraries nifh glutath ione S-transferase-LIM3 of Enigma indicated specific binding to Gly-Pr o-Hyd-Gly-Pro-Hyd-Tyr-Ala corresponding to the major endocytic code of InsR Peptide competition demonstrated that both Pro and Tyr residues were required for specific interaction of InsR with Enigma, In contras t to LIM3 of Enigma binding to InsR, LIM2 of Enigma associated specifi cally with the receptor tyrosine kinase, Ret. Ret was specific for LIM 2 of Enigma and did not bind other LIM domains tested, Mutational anal ysis indicated that the residues responsible for binding to Enigma wer e localized to the carboxyl-terminal 61 amino acids of Ret, A peptide corresponding to the carboxyl-terminal 20 amino acids of Ret dissociat ed Enigma and Ret complexes, while a mutant that changed Asn-Lys-Leu-T yr in the peptide to Ala-Lys-Leu-Ala or a peptide corresponding to exo n16 of InsR failed to disrupt the complexes, indicating the Asn-Lys-Le u-Tyr sequence of Ret is essential to the recognition moth for LIM2 of Enigma, We conclude that LIM domains of Enigma recognize tyrosine-con taining motifs with specificity residing in both the LIM domains and i n the target structures.