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Identification of structural characteristics that contribute to a difference in antiapoptotic function between human
H Chen1, G C Yan, M L Gishizky
1SUGEN, Inc., South San Francisco, California 94080, USA.
Abstract:
To determine whether potentiation of cell survival is an intrinsic function among the insulin receptor tyrosine kinase (RTK) family, we compared the ability of insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF-IR) cytoplasmic tails to attenuate apoptosis. Expression and activation of IGF-IR, in interleukin-3 (IL-3)-dependent 32Dcl.3 cells, prevents death under conditions of IL-3 withdrawal. In contrast, a chimeric receptor comprising the extracellular portion of IGF-IR fused to the cytoplasmic tail of IR (chIR) fails to promote cell survival when activated with ligand. Both chIR and IGF-IR exhibit comparable levels of enzymatic activity as evidenced by their ability to autophosphorylate and transphosphorylate the shc protein in vivo. Both chIR and IGF-IR can activate the MAPK signal transduction pathway; however, neither RTK is capable of promoting mitogenesis in the absence of IRS proteins. Structure function analysis of the IR cytoplasmic tail reveals that replacing the COOH-terminal 94 amino acids of the IR cytoplasmic tail with the comparable sequence from IGF-IR confers full antiapoptotic function. Furthermore, mutation of only two amino acids within IR, Phe-1264 and His-1265 to tyrosine (chIR/YY) is sufficient to impart a cell survival activity comparable to wild-type IGF-IR. Amino acid residues Phe-1264 and His-1265 of IR are in a region comparable to Tyr-1250 and Tyr-1251 within human IGF-IR. The amino acid sequence of IR from other species contains at least one tyrosine residue in this region, suggesting that differences in antiapoptotic function we observed may represent a characteristic unique to human members of this RTK family. The ability of IGF-IR or chIR/YY to prevent apoptosis is not blocked by addition of the PI3K inhibitor wortmannin. These studies define a critical region responsible for mediating cell survival through a novel interaction that is independent of mitogenesis.
Insights
Potentiating cell survival is an intrinsic function of the insulin-like growth factor 1 receptor (IGF-IR) but not the human insulin receptor (IR). Specific amino acid changes in the IR cytoplasmic tail confer anti-apoptotic activity.
Area of Science:
- Cellular Biology
- Molecular Biology
- Signal Transduction
Background:
- The insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF-IR) are receptor tyrosine kinases (RTKs) involved in cell growth and survival.
- Understanding the intrinsic functions of RTK family members is crucial for deciphering cellular signaling pathways.
Purpose of the Study:
- To investigate whether cell survival potentiation is an intrinsic function of the IR/IGF-IR tyrosine kinase (RTK) family.
- To identify the specific regions within the cytoplasmic tails of IR and IGF-IR responsible for mediating anti-apoptotic effects.
Main Methods:
- Utilized chimeric receptors combining extracellular domains of IGF-IR with cytoplasmic tails of IR (chIR) and vice versa.
- Assessed cell survival in IL-3-dependent 32Dcl.3 cells under IL-3 withdrawal conditions.
- Analyzed receptor activation, autophosphorylation, shc protein phosphorylation, and MAPK pathway activation.
- Performed structure-function analysis by mutating specific amino acids in the IR cytoplasmic tail.
Main Results:
- IGF-IR activation prevented apoptosis in IL-3-deprived cells, while chIR did not, despite comparable enzymatic activity and MAPK pathway activation.
- Replacing the C-terminal 94 amino acids of the IR cytoplasmic tail with the IGF-IR sequence conferred anti-apoptotic function.
- Mutation of two specific amino acids (Phe-1264 and His-1265) in the IR tail to tyrosine (chIR/YY) restored cell survival activity, mimicking IGF-IR.
- The anti-apoptotic function of IGF-IR and chIR/YY was independent of PI3K signaling, as it was not inhibited by wortmannin.
Conclusions:
- A critical region within the IR cytoplasmic tail, specifically amino acids Phe-1264 and His-1265, is responsible for mediating cell survival.
- These findings suggest that the potentiation of cell survival by this RTK region is independent of mitogenesis and may be a characteristic unique to human IR family members.
- The identified mechanism involves a novel interaction pathway distinct from PI3K signaling.