DISCUSSION

Bok: Could you clarify one thing? Was the central versus peripheral P216L e¡ect transient, and did it spread to the periphery over time as the frog got older? Or was it persistent?

Molday: The animals that we analysed were quite young . on average 4^8 weeks old. For P216L we only examined 4 week-old animals and we only saw degeneration in the central retina. We anticipate that the degeneration will spread to the peripheral region of the retina as the animals get older. Unfortunately, in this kind of experiment you have to sacri¢ce the animal. We did not generate enough transgenic X. laevis tadpoles to examine the temporal and spatial pro¢le for degeneration.

Bok: Do you have a line going or will you have to make new animals?

Molday: We didn’t make a line. We hope to generate additional mutant tadpoles and make a line in the future to examine the time dependence of degeneration.

LaVail: I want to make sure I understood you. You said you went up to about eight weeks in all of them. Would you think that with these kinds of changes piling up in the inner segment, you would ultimately get some changes in photoreceptors?

Molday: We did look at some of the animals up to 8 weeks. The wild-type transgenic animals looked quite normal, although we did not carry out a detailed study. I would suspect that the accumulation of the C214S dimeric species in the inner segment over time would cause problems, eventually leading to degeneration. In people with the C214S mutation, however, I would predict that photoreceptor degeneration is principally caused by low levels of wild-type peripherin.

McInnes: Have you seen any interaction between either of these proteins and ABCR?

Molday: No. We carried out extensive immunprecipitation and cross-linking studies and ¢nd no co-precipitation or cross-linking of ABCR with peripherin 2 or ROM1. This is a negative experiment. It is possible that detergent solubilization could disrupt existing weak ABCR^peripherin 2 interactions and cross-linking could be ine¡ective. At the present time, however, we have no evidence to suggest that ABCR strongly interacts with peripherin 2.

McInnes: We observed larger disks in the Rom1 mice, and Dean Bok has pointed out that the Rds heterozygote mice have large disks as well. How do you think that phenotype ¢ts into the biochemical model you are proposing?

Molday: It ¢ts quite well. Transgenic Xenopus tadpoles expressing wild-type transgene against a wild-type background express peripherin in excess over normal levels. At high transgenic peripherin expression we have seen a narrowing of the outer segment and smaller disks. This appears to indicate that excess peripherin 2 increases the rate of disk closure resulting in smaller disks.

When peripherin or ROM1 is in lower than normal amounts such as in the Rom1 knockout mouse and Rds heterozygote mouse, disk rim closure is slow resulting in larger disks. Interestingly, amphibians such as X.laevis with large disks do not have ROM1. This implies that ROM1 may be a negative regulator of disk size via its interaction with peripherin 2.

Hauswirth: In the peripherin mutations in which you see accumulation in the inner segment, have you stained with antibodies against the normal Xenopus proteins such as rhodopsin, to see whether there is some e¡ect on general transport to the outer segments?

Molday: We have examined endogenous wild-type peripherin 2 in the C214S transgenics that accumulate the mutant in the inner segment. In this instance, the tra⁄cking of endogenous wild-type peripherin 2 to the outer segment is not a¡ected by the presence of C214S accumulation in the inner segments. We have not looked at other outer segment proteins. Since outer segments appear normal in these animals, we assume that the tra⁄cking of other outer segment proteins is normal. However, this is a reasonable experiment to do. We should examine protein tra⁄cking to outer segments in these mutant animals by immuno£uorescence microscopy.

Daiger: There are three di¡erent polymorphic amino acid substitutions in human peripherin 304, 310 and 338, which lead to four di¡erent protein haplotypes. Have you looked for functional di¡erences in those four haplotypes? And in your constructs for dealing with human mutations, do you pick only one of those four haplotype backgrounds?

Molday: Our studies have concentrated on disease-linked mutations. We have not examined the polymorphic amino acid substitutions. We have no evidence that these changes will a¡ect the biochemical properties or subunit assembly of peripherin 2.

Dryja: There is no evidence that those polymorphisms are related to any human photoreceptor disease.

Daiger: No. I’m just asking whether the four di¡erent proteins produced by that locus are functionally relevant.

Dryja: One piece of evidence in favour of them not being relevant is that the £uorescent tag is placed on the end of the protein that has the polymorphic residues. Even with the tag on that end of the protein, the protein seems to work ¢ne. It looks like that end of the protein is not functionally important.

Bok: Xenopus actually has three di¡erent peripherin genes, leading to three di¡erent peripherin proteins in the outer segment.

Molday: We have concentrated on the major form that is most similar to human peripherin 2. I believe the two other isoforms are present in lower concentrations.

Bok: All three of the isoforms of peripherin/rds in Xenopus are readily detectable by immunocytochemistry, so they must be fairly abundant.

Dryja: I’d like to follow up this question about a possible interaction between ABCR and peripherin. One blot showed no evidence of an interaction, but if there were some way that the peripherin mutations interfered with ABCR activity, it might explain why we get these macular degeneration phenotypes with subretinal deposits that Alan Bird was mentioning that are reminiscent of the macular degeneration that is associated with ABCR mutations.

Molday: This would require a co-expression system in which one could coexpress wild-type and mutant peripherin with wild-type ABCR and look for di¡erences in the function of ABCR at a biochemical level. A system could be developed to examine the ATPase activity of ABCR in the presence of wild-type and mutant peripherin. This has not yet been done.

Travis: Benjamin Kaupp showed an interaction between free GARP and ABCR. Although there may not be a direct interaction between Rds and ABCR, they may both be talking through GARP.

Molday: The study by Kaupp and co-workers showing an interaction between ABCR and free GARP has been retracted (see the recent review article Kaupp & Seifert 2002). It would appear that the interaction they initially described was due to non-speci¢c interactions.

Nathans: I have two questions that might be related. What do you think controls the generation of incisures? You can imagine with this linkage to the plasma membrane how peripherin/rds complexes might assemble, but what makes them move into the interior of a disk to form an incisure, and what controls the number of incisures? And what is free GARP doing?

Molday: It seems that the number of incisures is often greater in larger animals. I do not know what the molecular or cellular mechanisms that control the number and formation of incisures. This, along with the molecular basis of disk formation, is poorly understood at the present time.

I can speculate on the possible role of free GARP. We know that GARP on the channel and free GARP bind to the peripherin/ROM1 complex at the rim region of disk membranes. The interaction of the GARP part of the B-subunit of the channel may be largely responsible for de¢ning the spatial relations of the disks to the plasma membrane via this protein^protein interaction and may represent ¢lamentous connections observed between the disk rim and plasma membrane by electron microscopy. One can suggest that free GARP may control the spatial arrangement of the stack of disks. If GARP forms a multi-subunit complex, then this complex can be envisioned to form a bridge between adjacent disks via interactions with the peripherin/ROM1 complex at the rim regions of disks. Therefore, GARP with a high content of proline residues would bind peripherin/ ROM1 and link together adjacent disk rims and disks and plasma membrane via protein^protein interactions involving peripherin^GARP complexes.

Nathans: Has free GARP been immunolocalized?

Molday: Yes. GARP has been localized along the rim regions of disk membranes and between the disks and plasma membrane using both post-embedding and preembedding immunogold labelling techniques.

Bok: You showed a transmission electron micrograph that displayed ¢laments going from neck to neck in the outer segment disk loops. Do you think this is free GARP?

Molday: That’s our speculation. Other proteins may also be involved although the number of unidenti¢ed candidate proteins in outer segments that are of su⁄cient quantity to represent the ¢laments observed between two disks and between a disk and plasma membrane is diminishing.

Aguirre: If I extend the free GARP question, in RPGR exon ORF15 there is a very glutamic acid-rich region which is almost GARP-like. Can you foresee a GARP-like role for RPGR in that region of the outer segment?

Molday: The GARP nomenclature is a bit of a misnomer. When GARP1 was identi¢ed it was characterized as having a string of glutamic acids near the C- terminal part of the protein. This is also present in the GARP part of the channel. GARP2, a truncated form of GARP1, however, is the predominant form in the outer segments and does not have this glutamic acid-rich region. In fact, it is a proline-rich protein as opposed to a glutamic acid-rich protein. My speculation would be that this glutamic acid-rich region, which is not well conserved, probably functions to extend the functional GARP domain further out thereby facilitating the interaction between the disc and the plasma membrane.

Reference

Kaupp UB, Seifert R 2002 Cyclic nucleotide-gated ion channels. Physiol Rev 82:769^824