Purification and Biochemical Properties of Rac1, 2, 3 and the Splice Variant Rac1b

Abstract

Rac proteins (Rac1, 1b, 2, 3) belong to the GTP-binding proteins (or GTPases) of the Ras superfamily and thus act as molecular switches cycling between an active GTP-bound and an inactive GDP-bound form through nucleotide exchange and hydrolysis. Like most other GTPases, these proteins adopt different conformations depending on the bound nucleotide, the main differences lying in the conformation of two short and flexible loop structures designated as the switch I and switch II region. The three distinct mammalian Rac isoforms, Rac1, 2 and 3, share a very high sequence identity (up to 90%), with Rac1b being an alternative splice variant of Rac1 with a 19 amino acid insertion in vicinity to the switch II region. We have demonstrated that Rac1 and Rac3 are very closely related with respect to their biochemical properties, such as effector interaction, nucleotide binding, and hydrolysis. In contrast, Rac2 displays a slower nucleotide association and is more efficiently activated by the Rac-GEF Tiam1. Modeling and normal mode analysis corroborate the hypothesis that the altered molecular dynamics of Rac2, in particular at the switch I region, may be responsible for different biochemical properties. On the other hand, our structural and biochemical analysis of Rac1b has shown that, compared with Rac1, Rac1b has an accelerated GEF-independent GDP/GTP-exchange and an impaired GTP-hydrolysis, accounting for a self-activating GTPase. This chapter discusses the use of fluorescence spectroscopic methods, allowing real-time monitoring of the interaction of nucleotides, regulators, and effectors with the Rac proteins at submicromolar concentrations and quantification of the kinetic and equilibrium constants.

Deciphering the Molecular and Functional Basis of Dbl Family Proteins

“…The intermediate efficiency group consists of the Dbl members p190 and p115 (29) as well as the oligo-specific Dbl proteins PRex1, Dbl, and Dbs. We also indexed Tiam1-Rac2 to this group as Tiam1 clearly revealed lower specificity for Rac1 and Rac3, as discussed previously (45,46). The third group with low efficiency is populated not only by Cdc42-specific hPem2/Collybistin and Tuba, Rac-specific TrioN, but also by PRex1, Dbl, Dbs, Vav2, and TrioN with broad specificity.…”

Specificity and Mechanism of Action of EHT 1864, a Novel Small Molecule Inhibitor of Rac Family Small GTPases

“…Rac1b, an alternative splice variant of Rac1, contains a 19-amino acid insert immediately behind the switch II region (residues 60 -76) of Rac1. This has been shown to be sufficient to disrupt effector interaction itself (19,30,57,58 Previous studies demonstrated that Ras causes activation of Rac and that Rac function is critical for Rasmediated growth transformation. In these studies, Rac function was blocked by use of a dominant negative Rac1(17N) mutant (11,12), which blocks RacGEF activation of Rac (59), or by NSC23766, a cellpermeable small molecule that can bind directly to Rac1 and prevent its activation by Rac-specific RhoGEFs (27).…”

“…Rac proteins were prepared similarly as described elsewhere (30,35,36). Briefly, BL21(DE3) cells expressing Rac1, Rac1b, Rac2, and Cdc42, respectively, were incubated at 37°C, before induction with 1 mM isopropyl 1-thio-␤-D-galactopyranoside for 4 h at 37°C (Rac3 required overnight induction at room temperature).…”

Regulation of NADPH Oxidase in Vascular Endothelium: The Role of Phospholipases, Protein Kinases, and Cytoskeletal Proteins

“…Rac belongs to the superfamily of Ras GTPases, and four isoforms, Rac1, Rac1b, Rac2, and Rac3, have been identified (87). Rac1 is expressed in nonhematopoietic cells and is likely the activator of Nox1, Nox3, Nox4, and Nox5, whereas Rac2 is expressed mostly in hematopoietic cells and is required for the activation of Nox2 in neutrophils (100).…”