Expression, Function and Trafficking of the Human ABCG2 Multidrug Transporter Containing Mutations in an Unstructured...

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• The human ABCG2 transporter is vital for xenobiotic and endobiotic transport, influencing drug resistance and gout.
• A specific cytoplasmic region (amino acids 354-367) of ABCG2, including lysines (K357-360) and threonine (T362), is implicated in protein function and cellular fate.
• Understanding this region's role is crucial for deciphering ABCG2's complex functions.

Purpose of the Study:

• To investigate the functional and trafficking effects of variants within the structurally unresolved cytoplasmic region of human ABCG2.
• To analyze the impact of the K360del variant and alanine replacements of neighboring lysines on ABCG2.
• To re-evaluate the role of T362 phosphorylation in ABCG2 function and expression.

Main Methods:

• Analysis of naturally occurring and engineered variants in human cells.
• Assessment of ABCG2 plasma membrane expression and cellular trafficking.
• Measurement of ABCG2 transport function and localization in polarized cells.
• Molecular dynamics simulations to study protein structural dynamics.

Main Results:

• The K360del variant significantly increased ABCG2 plasma membrane expression and accelerated trafficking.
• Alanine substitutions of neighboring lysines (K357, K358, K359) did not affect transport function or apical localization.
• T362A and T362E variants showed no measurable impact on ABCG2 function or expression, contrary to previous suggestions.
• Molecular dynamics simulations revealed increased mobility of mutant variants without altering the core protein structure.

Conclusions:

• The K360del variant enhances ABCG2 trafficking and cell surface expression.
• The unstructured cytoplasmic loop (a.a. 354-367) plays a role in regulating ABCG2 trafficking.
• The phosphorylation status of T362 does not appear to significantly influence ABCG2 function or expression.
• These findings contribute to understanding the regulatory mechanisms of ABCG2 function.