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Differential requirement for type I and type II transforming growth factor beta receptor kinase activity in
R A Anders1, J J Doré, S L Arline
1Thoracic Research Unit and Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, USA.
Abstract:
Transforming growth factor beta (TGFbeta) superfamily polypeptides regulate cell growth and differentiation by binding to single pass serine/threonine kinases referred to as TGFbeta type I and type II receptors. Signal propagation is dependent upon heteromeric (type I-type II) complex formation and transphosphorylation of the type I receptor by the type II receptor. While many of the phosphorylation events necessary for receptor signaling have recently been characterized, the role of TGFbeta receptor kinase activity in modulating receptor endocytosis has not been addressed. To that end, we have used chimeric receptors consisting of the extracellular domain of the granulocyte/macrophage colony-stimulating factor alpha and beta receptors spliced to the TGFbeta type I and type II transmembrane and cytoplasmic domains to address the specific role of type I and/or type II receptor kinase activity in TGFbeta receptor internalization, down-regulation, and signaling. To inactivate chimeric receptor kinase activity, point mutations in the ATP binding site were made at amino acids 232 and 277 in the type I and type II receptor, respectively. Either of these mutations abolished plasminogen activator inhibitor 1 protein expression stimulated by granulocyte/macrophage colony-stimulating factor activation of chimeric heteromeric type I-type II TGFbeta receptors. They did not, however, modulate TGFbeta signaling stimulated through the endogenous TGFbeta receptor. Although TGFbeta receptor signaling was dependent upon the kinase activity of both chimeric receptors, the initial endocytic response was distinctly regulated by type I and/or type II receptor kinase activity. For instance, while heteromeric receptor complexes containing a kinase-inactive type I receptor were endocytosed similarly to wild type complexes, the kinase activity of the type II TGFbeta receptor was necessary for optimal internalization and receptor down-regulation. Furthermore, these responses were shown to occur independently of type II receptor autophosphorylation but require a type II receptor capable of transphosphorylation.
Insights
Transforming growth factor beta (TGFbeta) receptor signaling involves complex formation and kinase activity. The type II TGFbeta receptor kinase activity is crucial for optimal receptor internalization and down-regulation, independent of autophosphorylation but requiring transphosphorylation capability.
Area of Science:
- Cell biology
- Molecular signaling
- Receptor biology
Background:
- Transforming growth factor beta (TGFbeta) superfamily regulates cell growth and differentiation via TGFbeta type I and type II receptors.
- Receptor signaling depends on heteromeric complex formation and transphosphorylation.
- The role of TGFbeta receptor kinase activity in receptor endocytosis remains unclear.
Purpose of the Study:
- Investigate the specific role of type I and/or type II TGFbeta receptor kinase activity in receptor internalization, down-regulation, and signaling.
- Utilize chimeric receptors to dissect the kinase-dependent mechanisms of TGFbeta receptor trafficking and signaling.
Main Methods:
- Constructed chimeric receptors by fusing extracellular domains of GM-CSF receptors with TGFbeta receptor transmembrane and cytoplasmic domains.
- Introduced point mutations in the ATP binding sites of type I (amino acid 232) and type II (amino acid 277) receptors to inactivate kinase activity.
- Assessed plasminogen activator inhibitor 1 (PAI-1) protein expression and TGFbeta signaling in response to chimeric receptor activation.
Main Results:
- Kinase activity of both type I and type II TGFbeta receptors is essential for TGFbeta receptor signaling.
- Type II TGFbeta receptor kinase activity is necessary for optimal receptor internalization and down-regulation.
- Receptor internalization and down-regulation are independent of type II receptor autophosphorylation but require transphosphorylation capability.
Conclusions:
- TGFbeta receptor signaling is dependent on the kinase activity of both receptor types.
- Type II TGFbeta receptor kinase activity plays a distinct and critical role in regulating receptor endocytosis and down-regulation.
- The kinase activity of the type II receptor, specifically its transphosphorylation ability, is a key determinant of TGFbeta receptor trafficking.