We investigated the role of the I-E-N-K-L (amino acids 1057-1061) sequence amino-terminal to Tyr1062 in Ret for binding of the Shc phosphotyrosine-binding (PTB) domain. Substitution of Ser for Ile1057 (I1057S), Ala for Asn1059 (N1059A), or Pro for Leu1061 (L1061P) in this sequence significantly decreased the transforming activity of Ret with the multiple endocrine neoplasm type 2A (MEN2A) mutation as well as the binding affinity of Shc to it in vivo and in vitro, indicating that these three amino acids play a role in Shc binding. In addition, as the RET protooncogene is translated as three isoforms of 1114 amino acids (Ret 51), 1106 amino acids (Ret 43), and 1072 amino acids (Ret 9) that differ from one another in the sequence carboxyl-terminal to Tyr1062, we examined whether these sequence differences influence the binding affinity of Shc to Ret. As a result, we found that the transforming activity of Ret 43 isoform with the MEN2A mutation and the binding affinity of Shc to it were very low, although the consensus sequence for the binding of the Shc PTB domain is conserved in the Ret 43 isoform. This finding suggested that the sequence carboxyl-terminal to Tyr1062 in Ret could also influence the binding affinity to Shc.