ARF GTPases and their GEFs and GAPs: concepts and challenges

Abstract

Detailed structural, biochemical, cell biological, and genetic studies of any gene/protein are required to develop models of its actions in cells. Studying a protein family in the aggregate yields additional information, as one can include analyses of their coevolution, acquisition or loss of functionalities, structural pliability, and the emergence of shared or variations in molecular mechanisms. An even richer understanding of cell biology can be achieved through evaluating functionally linked protein families. In this review, we summarize current knowledge of three protein families: the ARF GTPases, the guanine nucleotide exchange factors (ARF GEFs) that activate them, and the GTPase-activating proteins (ARF GAPs) that have the ability to both propagate and terminate signaling. However, despite decades of scrutiny, our understanding of how these essential proteins function in cells remains fragmentary. We believe that the inherent complexity of ARF signaling and its regulation by GEFs and GAPs will require the concerted effort of many laboratories working together, ideally within a consortium to optimally pool information and resources. The collaborative study of these three functionally connected families (≥70 mammalian genes) will yield transformative insights into regulation of cell signaling.

FIGURE 1:

Subcellular localization of the ARF family GTPases, ARF GEFs, and ARF GAPs. A schematic cell with organelles (in red) showing the localization of the GTPases (in light blue), GEFs (in purple), and GAPs (in green). More detailed information for these localizations is provided in references cited in the text.

FIGURE 2:

Structural determinants of ARF association with membranes and interactors. ARFs have four regions that change conformation between GDP- and GTP-bound forms: the canonical switch 1 (in orange) and switch 2 (in magenta) that directly sense the nature of the bound nucleotide; the myristoylated N-terminal helix (in blue), which is autoinhibitory in ARF-GDP and binds the membrane in ARF-GTP; and the interswitch (in red) that functions as a push button to ensure allosteric communication between the membrane- and the nucleotide-binding sites. GEFs, GAPs, and effectors generally bind to switch 1, switch 2, and/or the interswitch by one domain (in light yellow) and carry other domains that bind to the membrane (in light blue). The membrane bilayer is denoted in gray.

FIGURE 3:

Domain organization of ARF GEFs and ARF GAPs. A schematic of the domains present in each subfamily of the ARF GEFs (A) and ARF GAPs (B). The defining ARF GEF/Sec7 domain and the ARF GAP domain are aligned. Protein lengths are not drawn to scale. Abbreviations (in alphabetical order): A, ARF GAP lipid-packing sensor (ALPS); ANK, ankyrin repeat; BAR, Bin/Amphiphysin/Rvs; BoCCS, binder of coatomer, cargo, and SNARE; CALM BD, calm binding domain; CB, clathrin box; DCB, dimerization and cyclophilin binding; E/DLPPKP₈, 8 repeats of this primary sequence (single letter code); F-BOX, cyclin F protein interaction motif; FG repeats, multiple copies of XXFG repeated; GLD, GTP binding protein–like domain; GRM, Glo3 regulatory motif; HDS(1-4), homology downstream of Sec7; HUS, homology upstream of Sec7; IQ, isoleucine/glutamine calmodulin-binding motif; PBS, Paxillin binding site; PH, pleckstrin homology; Pro-rich, proline rich; RA, Ras association; Rho GAP, Rho GTPase-activating protein; SAM, sterile α motif; SHD, Spa homology domain.

FIGURE 4:

Evolution of the ARF family and its regulators. (A) The timing of the emergence of the relevant protein subfamilies is shown mapped on a simplified tree of eukaryotes. The polygons, circles, and squares denote the latest point by which the ARF GTPases, GAPs, and GEFs must have evolved, respectively, with the names of the subfamilies given to the right. The names of the eukaryotic supergroups are in italics, while the relevant “reconstructed ancestor” discussed in the text are in bold and noted by a dashed line. (B) Overlay of ARF1-5 evolution with that of the nine ARF GAP subfamilies that possess multiple paralogues. ARF evolution is depicted in black and Arf GAP in gray, with duplications at the base of Holozoa and Vertebrata. Relevant evolutionary transitions are illustrated by dashed lines.