News and Publications

Membrane Protein Topogenesis

M. Friedlander, M. Ritter, S. Hanekamp, K. Kinder, S.F. Friedlander, S.M. Simon,* K. Philipson**

* The Rockefeller University, New York, NY
** University of California, Los Angeles, CA

We are studying the mechanism whereby proteins are asymmetrically integrated into cell membranes. In addition to membrane protein topogenesis at the molecular level, we are investigating 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 of which contains 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. On the basis of these observations, we proposed a mechanism for membrane integration of polytopic proteins that involves multiple internal topogenic signals. We later extended these studies to examine the question of how these topogenic sequences sequentially insert the entire protein into the membrane. In collaboration with S. Simon's group at The Rockefeller University in New York, we found that within a range of nascent peptide lengths, opsin targets and translocates as efficiently after translation as it does during translation. A signal recognition particle is required for both types of translocation.

TOPOLOGY OF THE SODIUM-CALCIUM EXCHANGER FROM CARDIAC MUSCLE AND PHOTORECEPTORS

In collaboration with K. Philipson's group at the 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. 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 eucaryotic 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 is disrupted, although subsequent internal topogenic signals can target the protein to the membrane of the endoplasmic reticulum. Functionally expressed exchanger is being studied by using ion exchange assays and 2-photon scanning laser confocal microscopy of live cell cultures and retinal explants.

Neovascular Eye Diseases

M. Friedlander, E. Aguilar, F. Barnett, D. Cheresh, M. Dorrell, R. Gariano, K. Kinder, G. Nemerow, A. Otani, M. Ritter, M. Scharer-Schuksz, P. Schimmel, W. Richardson,* M. Fruttiger*

* University College, London, England

Neovascularization is the most common pathologic change associated with eye diseases that result in catastrophic loss of vision. Diabetic retinopathy and age-related macular degeneration are the leading causes of visual loss due to proliferation of new blood vessels. Current treatment for these conditions involves extensive destruction of retinal tissue with laser photocoagulation. If a less destructive, and more effective, treatment could be developed, visual loss could be markedly reduced in these patients.

We are characterizing and testing antibody, peptide, and organic antagonists to the integrins a_vß₃ and a_vß₅. Using chorioallantoic membrane, corneal, and retinal models of angiogenesis, we defined 2 angiogenic pathways on the basis of the dependency of the pathways on these distinct a_v integrins. We also showed that antagonists specific to each of these integrins selectively inhibit one of these pathways and that such pathways are involved in human neovascular eye diseases, as indicated by immunohistochemical studies of human pathologic specimens.

We are characterizing the molecular mechanisms that underlie these integrin-dependent angiogenic pathways and a variety of antagonists that may be useful in the treatment of clinically important angiogenic eye diseases. The role of the tumor suppresser genes p53 and p21 and of matrix metalloproteinases in these integrin-mediated angiogenic pathways is also under investigation.

In collaboration with P. Schimmel, Department of Molecular Biology, we are investigating a carboxyl-terminal fragment of tryptophan plus RNA synthetase as an inhibitor of retinal angiogenesis. This fragment is produced as a recombinant protein in bacteria and, in collaboration with G. Nemerow, Department of Immunology, as a transgene product in modified adenoviral vectors.

Friedlander Website