News and Publications

Membrane Protein Topogenesis

* Rockefeller University, New York, New York
** University of California, Los Angeles, California

We are studying the mechanism whereby proteins are asymmetrically integrated into cell membranes. In addition to studies of membrane protein topogenesis at the molecular level, we are studying defects in protein processing and insertion that occur in several degenerative diseases of the eye.

Topogenesis of Rhodopsin

Polytopic membrane proteins span the lipid bilayer several times and have hydrophilic domains exposed alternately on one side or the other of the membrane. Opsin, the apoprotein of rhodopsin, is representative of the larger family of G protein-coupled receptors that have 7 transmembrane segments and 8 hydrophilic domains, 4 of which face the biosynthetic compartment of the cell and 4 of which are extracellular. By constructing a series of opsin mutants, each containing only a single transmembrane segment, we showed that opsin has at least 5 internal signal sequences, each of which also expresses a strong or weak stop-transfer sequence.

We recently extended these studies to examine how these topogenic sequences sequentially insert the entire protein into the membrane. In collaboration with S. Simon's group at Rockefeller University, New York City, we found that within a range of nascent peptide lengths, opsin targets and translocates as efficiently during translation as it does after translation and that a signal recognition particle is required for both types of targeting. Furthermore, we showed that the posttranslational targeting and translocation requires nucleotide triphosphates (GTP alone is sufficient to fully restore targeting) but not cytosolic proteins. The addition of ATP was not specifically required, and nonhydrolyzable analogs of ATP that blocked 90% of the ATPase activity also had no inhibitory effect on translocation.

Topology of the Sodium-Calcium Exchanger From Cardiac Muscle and Photoreceptors

In collaboration with K. Philipson's group, University of California, Los Angeles, we are investigating the topology of the cardiac sodium-calcium exchanger. On the basis of hydropathic analysis of the amino acid sequence, the exchanger is proposed to contain 12 hydrophobic segments, the first of which is a cleaved signal sequence. By using a variety of reporter domains (glycosylation sites, epitopes, and proteolytic cleavage sites), we are analyzing the topology of the exchanger both in vitro and in oocyte expression systems. A full-length cDNA clone from photoreceptors is being similarly analyzed.

The cardiac exchangers have a cleaved amino-terminal signal sequence. Because nearly all other polytopic eukaryotic membrane proteins do not have cleaved signal sequences, we are investigating the putative role of such a sequence in the insertion and targeting of these exchangers. Our results indicate that the native, cleaved amino-terminal signal sequence is not necessary for insertion of a functional exchanger into the cell membrane.

In contrast, the photoreceptor exchanger does not have a cleaved amino-terminal signal sequence. If the amino-terminal 65 amino acids are deleted, translocation of the amino terminus of the protein is disrupted, but the remainder of the exchanger is integrated into the membrane. We are using ion exchange assays and 2-photon scanning laser confocal microscopy of live cell cultures and retinal explants to study functionally expressed exchanger.

Publications

Kanner, E.M., Friedlander, M., Simon, S.M. Co-translational targeting and translocation of the amino terminus of opsin across the ER membrane requires GTP but not ATP. J. Biol. Chem. 278:7920, 2003.

Angiogenesis-Dependent Disease

Most diseases that cause catastrophic loss of vision do so as a result of abnormal growth of blood vessels. Pathologic retinal and choroidal neovascularization leads to visual loss in diabetic retinopathy and age-related macular degeneration, respectively. Similarly, tumors depend on a blood supply for their growth and use these new vessels as an avenue for metastasis. Blood vessels themselves can generate tumors (e.g., hemangiomas) when the growth and organization of vascular endothelial cells are not properly controlled. Our goal is to understand the mechanisms of ocular neovascularization in normal and pathologic situations.

We used a neonatal mouse retina model to identify regulators of developmental angiogenesis and understand endothelial guidance mechanisms. In addition, in a long-standing collaboration with D.A. Cheresh, Department of Immunology, we are using this system to evaluate the role of integrins in these processes. In other studies, we found that bone marrow-derived endothelial precursor cells specifically target retinal astrocytes, incorporate into new vessels, and, in a model for retinal degeneration, rescue and stabilize a degenerating retinal vasculature.

In collaboration with P. Schimmel, Department of Molecular Biology, we found that these precursor cells can also be transfected with a plasmid that encodes a secreted form of a newly discovered antiangiogenic agent, T2 (a fragment of tryptophan tRNA synthetase). Injection of transfected cells into the eyes in newborn mice results in significantly reduced retinal vascularization. Most recently, we discovered that the precursor cells have a profound neurotrophic effect when injected into eyes of mice with inherited retinal degeneration; not only is the vasculature rescued in these mice but photoreceptors and visual function are also preserved.

Glioblastoma multiforme is an incurable brain tumor that is usually fatal within 1 year after diagnosis. We are using gene therapy and a rat model of this disease to study the efficacy of an antiangiogenic approach in treating these tumors. Hemangiomas are endothelial tumors that proliferate rapidly and later involute spontaneously. We are using DNA microarrays to study changes in gene expression as hemangiomas progress. Our goal is to identify (1) new targets for therapy for these tumors and (2) novel regulators of angiogenesis. In collaboration with G.R. Nemerow, Department of Immunology, we used pseudotyped adenovirus to selectively target specific cell types in the retina; by using the appropriate fiber type, we can deliver transgenes to cells, such as photoreceptors, that ordinarily are not targeted by adenovirus.

Publications

Aguilar, E., Friedlander, M., Gariano, R.F. Endothelial proliferation in diabetic retinal microaneurysms. Arch. Ophthalmol. 121:740, 2003.

Anecortave Acetate Clinical Study Group (Slakter, J.S., Binder, K., Blumenkranz, M., DeSmet, M., Fish, G., Friedlander, M., Gitter, K., Godley, B., Ho, A., Hudson, H., Van Kujik, E., Lewis, M., Rosenfeld, P., Russell, S., Sabates, F., Schachat, A., Schmidt-Erfurth, U., Schwartz, S., Singerman, L., Soubrane, G., Yannuzzi, L., D'Amico, D.J., Regillo, C., Mieler, W., Schneebaum, C., Beasley, C., Ciardella, A., Orlock, D., Goldberg, M.F., Jerdan, J., Krueger, S., Luna, S., Robertson, S., Sullivan, E., Zilliox, P.) Anecortave acetate as monotherapy for the treatment of subfoveal lesions in patients with exudative age-related macular degeneration (ARMD): interim (month 6) analysis of clinical safety and efficacy. Retina 23:14, 2003.

Heckenlively, J.R., Hawes, N.L., Friedlander, M., Nusinowitz, S., Hurd, R., Davisson, M., Chang, B. Mouse model of subretinal neovascularization with choroidal anastomosis. Retina, in press.

Von Seggern, D.J., Aguilar, E., Kinder, K., Fleck, S.K., Gonzalez Armas, J.C., Stevenson, S.C., Ghazal, P., Nemerow, G.R., Friedlander, M. In vivo transduction of photoreceptors or ciliary body by intravitreal injection of pseudotyped adenoviral vectors. Mol. Ther. 7:27, 2003.