ARF-Like (ARL) Proteins - Ras Superfamily Small G Proteins: Biology and Mechanisms 2 - page 232

share 80 % identity. The human and mouse paralogs share only 31 % identity

within each species. For comparison, human ARL13B and ARL13A share with the
single Chlamydomonas ARL13 31 % and 25 % identity, respectively, and have
been found to be functionally homologous. These values are low among ARF
family members, though quite high considering the time involved in evolutionary
terms, and are skewed to lower values in large part as a result of the relatively low
sequence conservation in the C-terminal half of the protein, termed the C-terminal
domain or CTD.
The ARL13B protein is made up of 2 domains: the N-terminal canonical GTP
binding (G) domain and a C-terminal domain (CTD) that has no clear homology to
other proteins or domains (Sun et al. 2004 ; Caspary et al. 2007 ; Duldulao
et al. 2009 ; Cevik et al. 2010 ). The G domain possesses all the hallmarks of a
GTPase, including 4 consensus nucleotide-binding motifs and the nucleotide-
sensitive switch 1 and switch 2 loops that mediate interaction with effectors and
modulators (Joneson et al. 1996 ; Kuai and Kahn 2000 ). ARL13B must employ
highly unusual, possibly unique, mechanisms for achieving temporal regulation in
signaling due to the absence of the conserved glutamine in the second consensus
nucleotide-binding motif: WDVGG Q in ARFs, FDLGG G in ARL13B. This single
Q-to-G change is predicted to be critical to ARL13B acting as a GTPase because the
homologous Q in ARF (and RAS and heterotrimeric G

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) proteins directly controls
both intrinsic and GAP-stimulated GTP hydrolysis; the homologous Q is commonly
mutated to generate a dominant-activating GTPase. Thus, ARL13B was predicted
to have a very slow rate of inactivation, or to use a different mechanism of GTP
hydrolysis, or both (Miertzschke et al. 2013 ). There is a precedent: RAP1 also lacks
the catalytic Q, so uses a distinct mechanism of GTP hydrolysis (Brinkmann
et al. 2002 ; Daumke et al. 2004 ; Scrima et al. 2008 ).
Three different missense mutations have been found in patients with Joubert
syndrome (Lee et al. 1997 ; Cantagrel et al. 2008 ; Thomas et al. submitted). Two of
the 3 point mutants in ARL13B that cause Joubert syndrome are in switch 2, R79Q
and Y86C, while the third is in the CTD (R200C). Arl13b hnn mice are embryonic
lethal, yet Joubert patients reach adulthood with specific neural, ocular, and renal
defects, consistent with each mutation affecting a subset of Arl13b phenotypes
(Caspary et al. 2007 ; Parisi and Glass 2012 ). Thus, we expect that each of these
mutants is a hypomorph and affects a subset of ARL13B effectors. Such mutants
offer outstanding tools that often prove invaluable in dissecting roles for ARL13B
in cilia and cell biologies. Testing these mutations in model systems has confirmed
the importance of those residues to ARL13 functions across species (Duldulao
et al. 2009 ; Cevik et al. 2010 ). These three residues and mutations have been
analyzed structurally using the crystal structure of the ARL13B ortholog from
Chlamydomonas reinhardtii to generate a number of conclusions and predictions
regarding their effects on the protein's structure and functions (Miertzschke
et al. 2013 ). Finally, all three of these mutations are conserved in ARL13As,
making it likely that there is some level of functional redundancy between the
paralogs in each species that has two genes.

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