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Separate endocytic pathways of kinase-defective and -active EGF receptor mutants expressed in same cells
A M Honegger1, A Schmidt, A Ullrich
1Rorer Biotechnology, Inc., King of Prussia, Pennsylvania 19406.
Abstract:
Ligand binding to the membrane receptor for EGF induces its clustering and internalization. Both receptor and ligand are then degraded by lysosomal enzymes. A kinase defective point mutant (K721A) of EGF receptor undergoes internalization similarly to the wild-type receptor. However, while internalized EGF molecules bound to either the wild-type or mutant receptors are degraded, the K721A mutant receptor molecules recycle to the cell surface for reutilization. To investigate the mechanism of receptor trafficking, we have established transfected NIH-3T3 cells coexpressing the kinase-negative mutant (K721A) together with a mutant EGF receptor (CD63) with active kinase. CD63 was chosen because it behaves like wild-type EGF receptor with respect to biological responsiveness and cellular routing but afforded immunological distinction between kinase active and inactive mutants. Although expressed in the same cells, the two receptor mutants followed their separate endocytic itineraries. Like wild-type receptor, the CD63 mutant was downregulated and degraded in response to EFG while the kinase-negative mutant K721A returned to the cell surface for reutilization. Intracellular trafficking of EGF receptor must be determined by a sorting mechanism that specifically recognizes EGF receptor molecules according to their intrinsic kinase activity.
Insights
The kinase activity of epidermal growth factor (EGF) receptors determines their fate after internalization. Kinase-inactive EGF receptors are recycled, while active ones are degraded, revealing a novel sorting mechanism.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Epidermal growth factor (EGF) receptor signaling is crucial for cell growth and differentiation.
- Ligand binding triggers EGF receptor clustering, internalization, and lysosomal degradation.
- Kinase activity of the EGF receptor plays a role in its cellular trafficking.
Purpose of the Study:
- To investigate the mechanism of EGF receptor trafficking.
- To determine if EGF receptor kinase activity dictates its endocytic pathway and degradation.
- To elucidate the sorting mechanisms governing receptor fate.
Main Methods:
- Utilized NIH-3T3 cells coexpressing two distinct EGF receptor mutants: a kinase-defective mutant (K721A) and a kinase-active mutant (CD63).
- Employed immunological distinction to track separate receptor populations within the same cell.
- Analyzed receptor internalization, trafficking, degradation, and recycling pathways.
Main Results:
- Both wild-type and kinase-active EGF receptors (CD63 mutant) were internalized and degraded upon EGF binding.
- The kinase-defective EGF receptor (K721A mutant) was internalized but recycled back to the cell surface.
- Distinct endocytic itineraries were observed for active and inactive EGF receptors within the same cell.
Conclusions:
- EGF receptor trafficking is regulated by a sorting mechanism that distinguishes between kinase-active and kinase-inactive receptors.
- Intrinsic kinase activity is a critical determinant for the degradation or recycling of internalized EGF receptors.
- This finding reveals a novel layer of regulation in receptor-mediated signaling pathways.