Activated or dominant inhibitory mutants of Rap1A decrease the oxidative burst of Epstein-Barr virus-transformed...

Area of Science:

• Cellular Biology
• Immunology
• Biochemistry

Background:

• Rap1A, a Ras superfamily GTP-binding protein, is abundant in phagocyte membranes.
• Rap1A copurifies with cytochrome b558, a component of the NADPH oxidase complex.
• The role of Rap1A in regulating the oxidative burst in phagocytes and B lymphocytes is unclear.

Purpose of the Study:

• To investigate the involvement of Rap1A in the regulation of the NADPH oxidase complex and oxidative burst in human B lymphocytes.
• To determine if GTP-bound and GDP-bound states of Rap1A differentially affect NADPH oxidase activity.

Main Methods:

• Stable transfection of human Epstein-Barr virus-transformed B lymphocytes with Rap1A mutants (GTP-bound 63E and GDP-bound 17N).
• Assessment of phorbol ester-stimulated superoxide (O2-.) production.
• Evaluation of cell viability, proliferation, cell-surface marker expression, and interleukin-8 generation.

Main Results:

• Both Rap1A 17N (GDP-bound) and 63E (GTP-bound) mutants significantly inhibited phorbol ester-stimulated O2-. production (by 50% and 80%, respectively).
• Transfection with wild-type Rap1A cDNA had no effect on the respiratory burst.
• No adverse effects on cell viability, proliferation, cell-surface markers, or IL-8 generation were observed.

Conclusions:

• Rap1A acts as a regulator of O2-. formation in intact human B lymphocytes.
• The inhibitory effects of both GTP- and GDP-bound Rap1A mutants suggest Rap1A functions in a dynamic cycle, unlike the unidirectional pathway of Rac.
• These findings elucidate a novel regulatory mechanism for the NADPH oxidase complex.