Last week’s blog described the crystal structure of the C-terminal domain of FLCN, which was published in a recent paper by Nookala et al. (2012). FLCN was found to have a similar structure to that of DENN domain proteins, which function as Rab guanine nucleotide exchange factors (GEFs). As such, Nookala et al. proposed a novel function for FLCN as a Rab GEF. Here, we introduce Rab GTPases, GEFs and DENN domains and discuss the future research which may be used to confirm FLCN’s function.
Rabs are the largest family of small GTPases, with around 70 identified in humans. GTPases cycle between an active GTP-bound state and an inactive GDP-bound state, regulating many processes within the cell. Rab GTPases specifically regulate membrane trafficking. Molecules move around the cell within vesicles, which bud from one membrane before fusing with an organelle or membrane where the cargo is released. Rabs ensure that the vesicle, and hence its cargo, is delivered to the correct destination.
Rabs are activated by GEFs which facilitate the release of GDP, allowing GTP, which is present at much higher levels in the cell than GDP, to bind to the Rab. Several DENN GEFs have been shown to facilitate this exchange through a direct interaction with their associated Rab. GEFs are localised to specific areas in the cell, ensuring that Rabs are only activated at the correct location. GEFs can be classified by their distinct protein domains; however GEFs for most Rabs are yet to be identified.
DENN domain proteins are a class of Rab GEFs. Differentially expressed in normal cells and neoplasia (DENN) domain proteins were first identified due to their variable mRNA expression levels in tissues and cell lines (Chow and Lee, 1996). Prior to the identification of FLCN as a DENN protein, 18 other human DENN proteins had been identified. The DENN domain is poorly characterised but conserved throughout evolution. It consists of three regions: the upstream (u-DENN), core (c-DENN) and downstream (d-DENN) regions, which are separated by linkers of various lengths.
The first structure of a DENN domain was determined by Wu et al. (2011), who described the structure of DENN1B-S bound to Rab35. The c-DENN and d-DENN regions of DENN1B-S overlay almost exactly with the C-terminal domain of FLCN. The u-DENN region of DENN1B-S was found to contain a longin domain, a domain which is found in several proteins involved in membrane trafficking. Structure prediction programs suggest that the N-terminal of FLCN also contains a longin domain (Nookala et al., 2012), but it would be interesting to study this further as it would give support to FLCN’s proposed role in membrane trafficking.
The dysregulation of DENN proteins has been associated with diseases such as asthma, Alzheimer’s and several cancers. Additionally, DENND2B functions as a tumour suppressor. It is possible that mutations in FLCN cause a dysregulation of membrane trafficking which leads to the symptoms of BHD syndrome. It would be interesting to study vesicle transport in FLCN-null cells, as well as other processes that are regulated by small GTPases.
- Nookala RK, Langemeyer L, Pacitto A, Ochoa-Montano B, Donaldson JC, Blaszczyk BK, Chirgadze DY, Barr FA, Bazan JF, Blundell TL. 2012 Crystal structure of folliculin reveals a hidDENN function in genetically inherited renal cancer. Open Biol 2: 120071. http://dx.doi.org/10.1098/rsob.120071
- Wu X, Bradley MJ, Cai Y, Kümmel D, De La Cruz EM, Barr FA, Reinisch KM. Insights regarding guanine nucleotide exchange from the structure of a DENN-domain protein complexed with its Rab GTPase substrate. Proc Natl Acad Sci U S A. 2011 Nov 15;108(46):18672-7. PMID: 22065758
- Chow VT, Lee SS. DENN, a novel human gene differentially expressed in normal and neoplastic cells. DNA Seq. 1996;6(5):263-73. PMID: 8988362
- Marat AL, Dokainish H, McPherson PS. DENN domain proteins: regulators of Rab GTPases. J Biol Chem. 2011 Apr 22;286(16):13791-800. PMID: 21330364
- Yoshimura S, Gerondopoulos A, Linford A, Rigden DJ, Barr FA. Family-wide characterization of the DENN domain Rab GDP-GTP exchange factors. J Cell Biol. 2010 Oct 18;191(2):367-81. PMID: 20937701
www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.