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 DereticDepartment 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

BBRTCsTGNGolgiNucleus

CTS FR binds ASAP1

Rhodopsin LNKQ FR NCSstr3 FKQG FR R ISmo T L L I WR RTODR-10 I I RD FR RT

CTS : FR

ADRP Mutations

DCB HUS Sec7 HDS1 HDS2 HDS3

GBF1

VxPx

N-terminus

H-8FR

ASAP1Rhodopsin C-terminus

Arf4

Ct

ADRP Mutations

Rhodopsin

CTS : V x P x

SRV Q PQ I R V A PG

Polycystin 2CNGB 1b

SQV A P ANKV H PSSPolycystin 1

* * *

CTS VxPx binds Arf4

I5V

S7A I100V

L130M

I136VN150S

E168D

N175H

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.