1. The role of Arfs, the Arf Guanine Nucleotide Exchange Factor (GEF)
GBF1 and Rab6 in the transport of Rhodopsin in Photoreceptor Cells
Alesia Vialichka, Vasundhara Kandachar, Dusanka Deretic
Department of Surgery/Opthamalogy, University of New Mexico
Rhodopsin is a light sensitive protein, which is synthesized in the Golgi
and transported to the modified primary cilium called the Rod Outer
Segment of photoreceptor cells in the retina. The components involved
in the initiation of vesicular rhodopsin trafficking are not yet fully
understood. The importance of Arf4 in recognition and binding of
rhodopsin has been shown, yet GBF1 is only beginning to be considered
as an Arf4 Guanine Nucleotide Exchange Factor (GEF). Direct
interaction between Arf4 and GBF1has been noted previously. In
addition to the catalytic Sec7 domain, GBF1 contains a dimerization and
cyclophilin binding (DCB) domain, a homology upstream of Sec7 (HUS)
domain, and three homology downstream of Sec7 (HDS) domains. Here,
we used GST fusion proteins containing the functional domains of
GBF1. To differentiate the affinity for each domain, we employed GST-
DCB-HUS and GST-Sec7-HDS1. GBF1 GST fusion proteins were
incubated with recombinant Arfs pre-loaded with GTPγS or GDPβS, We
examined the interaction between two GBF1 domain segments, and
Arf1, 4, 5, 6, and Rab6, bound to GDP or GTP. In vitro, we show that
Arfs 1, 4, 5 and 6, and Rab6 interact with GBF1, but Arf4 does so with
highest affinity. Because Arf4 is specifically involved in rhodopsin
trafficking, our study implies that in different cell systems the high affinity
of GBF1 for Arf4 may be compensated by downregulating its expression
and upregulating the expression of other Arfs.
Eukaryotic cells have many specialized membrane compartments.
They are always exchanging proteins and lipids, and this is done through
vesicular transport. We are interested in how this vesicular transport is
utilized in photoreceptor cells. Specifically, we are studying the
mechanism of how the protein rhodopsin is packaged and transported in
photoreceptor cells of the retina.
The photoreceptor rod outer segment (ROS) is a sensory organelle
derived from a cilia. It is filled with membranous disks which house the
light receptor rhodopsin, a prototypic GPCR, and other necessary photo
transduction machinery.
Renewal of the ROS membrane is driven by post-TGN rhodopsin
transport carriers (RTCs). RTCs traffic components through the rod inner
segment (RIS) and fuse with the RIS plasma membrane. RTC budding is
controlled by the binding of a small GTPase Arf4 to the rhodopsin C-
terminal VxPx cilliary targeting signal. This initializes the assembly of the
Arf GAP ASAP1/Rab11/FIP3 targeting complex.
The cilliary targeting signals VxPx and FR are conserved among all
sensory receptors. The CTSs are sites of rhodopsin mutations which
cause blindness in Autosomal Dominant Retinitis Pigmentosa (ADRP).
ABSTRACT
BACKGROUND
Thank you to Toby Hurd for supplying the GST-Arf4 mutants, as well as the
sequence and mutagenesis charts.
Thank you to Catherine Jackson for supplying the purified Arf1.
We found that although Arf1, 4, 5, and 6 interact with GBF1, Arf4
has a 10x higher affinity for the DCB-HUS domain than Arf1 does.
This helps us understand the regulation of Arfs in the cell during
trafficking. Interestingly, Arf6 shows a higher affinity for the
catalytic Sec7-HDS1 domain, unlike the other Arfs illustrated.
Rab6 is also seen binding the DCB-HUS domain of GBF1.
This work was supported by the UNM NIH CTSA Grant UL1TR000041 and NIH NE1 Grant EY12421 to D. D.
GST pulldown analysis of interactions between GBF1 DCB-HUS and Sec7-HDS1 domains with Arf1,
4, 5, 6, and Rab6.
RESULTS
SUMMARY
REFERENCES
GST pulldown analysis of interactions between Arf4 mutants and
Rhodopsin.
Figure 1. The comparison of the pulldowns of Arfs and Rab6 by different GBF1 domains. Arf4 has
the highest affinity for GBF1. Arf4 has a 10 fold higher affinity for DCB-HUS1 than Arf1. Arf4 does not
discriminate between GDPβS and GTPγS bound DCB-HUS domain. Rab6, Arf1,4,5 have a stronger
preference towards the DCB-HUS domain, while Arf6 has a clear preference to the Sec7HDS1 domain of
GBF1. Protein-protein interaction was performed and analyzed through Western Blot.
Figure 2. Interaction between Rhodopsin and Arf4. Rhodopsin was not
pulled down by GST-Arf4 in this experiment. This indicates that although
Arf4 and rhodopsin do interact, this interaction is not stable enough for
this pulldown. Another component of the RTC complex needs to be
included to stabilize the binding of rhodopsin to Arf4.
Rod Outer
Segment
(ROS)
Rod Inner
Segment
(RIS)
Synaptic
terminal
Cilium
BB
RTCs
TGN
Golgi
Nucleus
CTS FR binds ASAP1
Rhodopsin LNKQFRNC
Sstr3 FKQGFRRI
Smo T L L I WRRT
ODR-10 I I RDFRRT
CTS : FR
ADRP Mutations
DCB HUS Sec7 HDS1 HDS2 HDS3
GBF1
N-terminus
H-8
FR
ASAP1
Rhodopsin C-terminus
Arf4
ADRP Mutations
Rhodopsin
CTS : V x Px
SRVQPQ
IRVAPG
Polycystin 2
CNGB 1b
SQVA PA
NKVHPSSPolycystin 1
* * *
CTS VxPx binds Arf4
I5V
S7A
I100V
L130
M
I136V
N150S
E168D
N175
H
GDP
The domain structure of GBF1.
The segments of GBF1 which were used in GST
pulldowns with their size as well as corresponding
domains.
Sec7 is a highly conserved
catalytic domain, while the
other domains are regulatory.
The preference of Arf and Rab
for either domain will also help
us understand their role in
rhodopsin trafficking.
We believe that adding ASAP1
will improve the pull down of
rhodopsin with Arf4 and detect
the difference between the
mutants.