Specificity of LIM domain interactions with receptor tyrosine kinases.
LIM domains, Cys-rich motifs containing approximately 50 amino acids found 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-hybrid system, and glutathione S-transferase fusions. Enigma contains three LIM domains within its carboxyl terminus and LIM3 of Enigma specifically recognizes active but not mutant endocytic codes of the insulin receptor (InsR) (Wu, R. Y., and Gill, G. N. (1994) J. Biol. Chem. 269, 25085-25090). Interaction of two random peptide libraries with glutathione S-transferase-LIM3 of Enigma indicated specific binding to Gly-Pro-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 contrast to LIM3 of Enigma binding to InsR, LIM2 of Enigma associated specifically with the receptor tyrosine kinase, Ret. Ret was specific for LIM2 of Enigma and did not bind other LIM domains tested. Mutational analysis indicated that the residues responsible for binding to Enigma were localized to the carboxyl-terminal 61 amino acids of Ret. A peptide corresponding to the carboxyl-terminal 20 amino acids of Ret dissociated Enigma and Ret complexes, while a mutant that changed Asn-Lys-Leu-Tyr in the peptide to Ala-Lys-Leu-Ala or a peptide corresponding to exon16 of InsR failed to disrupt the complexes, indicating the Asn-Lys-Leu-Tyr sequence of Ret is essential to the recognition motif for LIM2 of Enigma. We conclude that LIM domains of Enigma recognize tyrosine-containing motifs with specificity residing in both the LIM domains and in the target structures.
Wu
R
R
Department of Biology, University of California San Diego, La Jolla, California 92093-0650, USA.
Durick
K
K
Songyang
Z
Z
Cantley
L C
LC
Taylor
S S
SS
Gill
G N
GN
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D016384:Consensus Sequence; D003545:Cysteine; D004705:Endocytosis; D066246:ErbB Receptors / Q000378:metabolism; D005091:Exons; D005982:Glutathione Transferase; D008322:Mammals; D051379:Mice; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000096:biosynthesis / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D013379:Substrate Specificity; D014162:Transfection
D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D066246:ErbB Receptors; D020794:Receptor Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D003545:Cysteine