Abstract
Abstract: :
Purpose: In the vertebrate retina, all trans-retinoic acid (at-RA) acts as a light-dependent neuromodulator, in addition to its regulatory role in gene transcription. at-RA induces adaptational effects in retinal horizontal cells that underlie light adaptation, but the molecular mechanisms of its action are still unknown. Recent findings in fish retina (Zhang & McMahon, 2000, PNAS 97: 14754) suggest that at-RA binds to an external membrane binding site, which activates an intracellular signal transduction pathway. We therefore studied the distribution of retinoic acid receptor alpha-like (RARα-like) proteins in the carp retina, and the existence of putative at-RA-binding proteins in carp retinal membranes. Methods: In order to reveal specific at-RA-binding, a receptor binding assay with different retinal fractions of either cytosolic or membrane proteins was carried out using tritiated at-RA ([3H]at-RA) as ligand. Furthermore, a photo-affinity-labeling assay and SDS gel electrophoresis were used to label and separate [3H]at-RA-binding proteins in these fractions. The distribution of RARα-like proteins in the carp retina was analyzed by means of immunochemistry using a RARα antibody. Results: Specific binding of [3H]at-RA was measured in membrane fractions (KD = 37,7nM) as well as in cytosolic fractions (KD = 21,3nM) of the carp retina. Photo-affinity-labeling and autoradiography revealed the existence of several at-RA-binding proteins in the cytosolic fraction and 6 different at-RA-binding proteins in the membrane fraction. One of these proteins had a molecular weight of 43 kD and subsequent immunobiochemistry revealed a RARα-ir protein with the same molecular weight in the membrane fraction and in isolated horizontal cells. In retinal cryosections of the carp retina, application of the same antibody resulted in a prominent, punctate labeling of the outer plexiform layer and in addition, isolated horizontal cells were also strongly labeled. Conclusions: The carp retina reveals different at-RA-binding membrane proteins. One of these proteins co-migrates with a RARα-like protein enriched in membranes and horizontal cells, suggesting that horizontal cells express a membrane-bound at-RA-binding protein.
Keywords: retinoids/retinoid binding proteins • retina: distal(photoreceptors, horizontal cell • signal transduction