Abstract
Integrin beta1C is an alternatively spliced cytoplasmic variant of the beta1 subunit that potently inhibits cell cycle progression. In this study, we analyzed the requirements for growth suppression by beta1C. A chimera containing the extracellular/transmembrane domain of the Tac subunit of the human interleukin 2 receptor (gp55) fused to the cytoplasmic domain of beta1C (residues 732-805) strongly inhibited growth in mouse 10T1/2 cells even at low expression levels, whereas chimeras containing the beta1A, beta1B, beta1D, beta3, and beta5 cytoplasmic domains had weak and variable effects. The beta1C cytoplasmic domain is composed of a membrane proximal region (732-757) common to all beta1 variants and a COOH-terminal 48-amino acid domain (758-805) unique to beta1C. The beta1C-specific domain (758-805) was sufficient to block cell growth even when expressed as a soluble cytoplasmic green fluorescent protein fusion protein. These results indicate that growth inhibition by beta1C does not require the intact receptor and can function in the absence of membrane targeting. Analysis of deletions within the beta1C-specific domain showed that the 18-amino acid sequence 775-792 is both necessary and sufficient for maximal growth inhibition, although the 13 COOH-terminal residues (793-805) also had weak activity. Finally, beta1C is known to be induced in endothelial cells in response to tumor necrosis factor and is down-regulated in prostate epithelial cells after transformation. The green fluorescent protein/beta1C (758-805) chimera blocked growth in the human endothelial cell line EV304 and in the transformed prostate epithelial cell line DU145, consistent with a role for beta1C as a growth inhibitor in vivo.
Highlights
Protein interactions, intracellular ion concentrations, and lipid metabolism
The 1A cytoplasmic domain has been shown to interact with the cytoskeletal proteins ␣-actinin, filamin, and talin, and sequences within the unique 1A domain are required for focal adhesion localization (8 –15)
We found that expression of 1C induced a potent inhibition of cell cycle progression, leading to arrest in late G1 [16]. 1C failed to localize to focal adhesions, suggesting that it has altered interactions with cytoskeletal proteins [16]. 1C blocked the growth of Chinese hamster ovary cells [17]
Summary
Protein interactions, intracellular ion concentrations, and lipid metabolism. Integrin cytoplasmic domains lack intrinsic enzymatic activity but have been shown to activate a variety of signaling pathways including protein kinases (focal adhesion kinase, integrin-linked kinase, protein kinase C, and mitogenactivated protein kinase), lipid kinases, phospholipases, and small GTP-binding proteins (Ras, Rac, and Cdc 42) and to bind to cytoskeletal proteins including ␣-actinin, talin, filamin, and paxillin. The 1A cytoplasmic domain has been shown to interact with the cytoskeletal proteins ␣-actinin, filamin, and talin, and sequences within the unique 1A domain are required for focal adhesion localization (8 –15). Taken together, these observations suggest that alternative splicing within the 1 gene may have profound effects on receptor function. The functions of 1C in vivo are unknown, but 1C expression has been shown to correlate with growth arrest. We found that the 1C-specific domain blocks DNA synthesis in endothelial and prostate carcinoma cells, suggesting that 1C protein may inhibit growth in vivo
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