Brown, Anna Marie; (1997) Small GTP-binding proteins in the regulation of exocytosis in mast cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Permeabilised rat peritoneal mast cells can be stimulated to secrete by the addition of calcium and GTPγS. If. however, the cells are stimulated subsequent to permeabilisation. they become increasingly refractory to stimulation, being only able to secrete, for example, 30-40% of their contained hexosaminidase after 10 minutes compared to almost 100% when stimulated at the time of permeabilisation. This phenomenon is called "rundown" and is thought to be due to the loss of cytosolic proteins involved in exocytotic regulation which leak from the cell through the membrane lesions created by the permeabilising agent. Using the recovery from rundown as the basis of a bioassay, I have collaborated on the purification of two such cytosolic factors from bovine brain cytosol which appear to act as exocytotic regulators. One factor was frilly purified and identified as a complex of Rac1 (a small GTP-binding protein of the Rho family) and Rho GDI. I found that the purified complex retards rundown, whilst Rho GDI accelerates rundown when applied alone. Rac2 is understood to be the predominant form of Rac expressed in myeloid cells. I have purified recombinant Rac2 from E.Coli and found that it retards rundown when preactivated by binding GTPγS before application to the cells, confirming that Rac can play a role in mast cell secretion. In addition, I found that a dominant negative mutant form of Rac2 inhibits secretion induced by GTPγS. Cdc42. another Rho-related GTPase which can interact with some Rac effectors, was also purified from E.Coli. Preactivated Cdc42 was found to retard rundown, but the concentration-effect relationship indicates that Cdc42 interacts with more than one effector. Also, a dominant negative mutant Cdc42 inhibits GTPγS-induced secretion. By introducing both Rac2 and Cdc42 simultaneously into permeabilised cells. I have obtained evidence suggesting that Cdc42 can interact with the Rac2 effector in addition to a second (possibly specific) effector.

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