Localization Versus Function of Rab3 Proteins

Abstract

Rab3A, Rab3B, Rab3C, and Rab3D constitute a family of GTP-binding proteins that are implicated in regulated exocytosis. Various localizations and distinct functions have been proposed for different and occasionally even for the same Rab3 protein. This is exemplified by studies demonstrating that deletion of Rab3A in knock-out mice results in dysregulation of the final stages of exocytosis, whereas overexpression of Rab3A in neuroendocrine cells causes nearly complete inhibition of Ca 2؉-triggered exocytosis. We have now examined the properties of all Rab3 proteins in the same assays, with the long-term goal of identifying a common conceptual framework for their functions. Using quantitative immunoblotting, we found that all four Rab3 proteins were expressed in brain and endocrine tissues, although at widely different levels. Rab3A, Rab3B, and Rab3C co-localized to synaptic and secretory vesicles consistent with potential redundancy, whereas Rab3D was expressed at high levels only in the endocrine pituitary (where it was more abundant than Rab3A, Rab3B, and Rab3C combined), in exocrine glands, and in adipose tissue. In transfected PC12 cells, all four Rab3 proteins strongly inhibited Ca 2؉ -triggered exocytosis. Except for a mutation that fixes Rab3 into a permanently GDP-bound state, all Rab3 mutations tested had no effect on this inhibition, including a mutation in the calmodulin-binding site that was described as inactivating (Coppola, T., Perret-Menoud, V., Lü thi, S., Farnsworth, C. C., Glomset, J. A., and Regazzi, R. (1999) EMBO J. 18, 5885-5891). Unexpectedly, overexpression of wild type Rab3A and permanently GTP-bound mutant Rab3A in PC12 cells caused a loss of secretory vesicles and an increase in constitutive, Ca 2؉ -independent exocytosis that correlated with the inhibition of regulated Ca 2؉ -triggered exocytosis. Our data indicate that overexpression of Rab3 in PC12 cells impairs the normal control of the final step in exocytosis, thereby converting the regulated secretory pathway into a constitutive pathway. These results offer an hypothesis that reconciles Rab3 transfection and knockout studies by suggesting that Rab3 functions as a gatekeeper of a late stage in exocytosis.

Annexin A2 Interactions with Rab14 in Alveolar Type II Cells

“…In mammalian cells, Rab proteins play a central role in constitutive and regulated exocytosis. Rab3A is one of the most notable and well characterized Rabs and is active in the regulated secretory pathway in the neuron (41). Involvement of Rab3A in insulin exocytosis was successively confirmed in vivo using Rab3A knockout mice (42).…”

Local Control of AMPA Receptor Trafficking at the Postsynaptic Terminal by a Small GTPase of the Rab Family

“…However, it is surprising that very few studies have addressed the role of Rab proteins in neuronal function, where polarized membrane traffic is essential to maintain presynaptic and postsynaptic function. The notable exception is Rab3, which is known to regulate neurotransmitter vesicle fusion and short-term plasticity at the presynaptic terminal (26), although the precise mechanisms underlying this regulation are still being elucidated (65,66).…”

Proteolytic Processing of Amyloid-β Precursor Protein by Secretases Does Not Require Cell Surface Transport

“…Constitutive hGH secretion was measured as described previously (36,45) and is expressed as fold-over cellular hGH. Immunocytochemistry of transfected HeLa cells was performed essentially as described by Cao and Sü dhof (9) using polyclonal syntaxin antibodies and monoclonal antibodies to GM130 (Transduction Laboratories) and goat anti-rabbit or goat anti-mouse secondary antibodies coupled with Alexa Fluor 488 and Alexa Fluor 546 (Molecular Probes).…”