Ligand-induced clustering of asialoglycoprotein receptors on rat hepatocytes at 4 degrees C

The platinum-carbon replica technique was applied to rat hepatocyte monolayers to visualize the distribution of the receptor responsible for the clearance of asialoglycoproteins. In a first series of experiments, hepatocytes were mildly fixed, either immediately after transfer to 4 degrees C, or after up to 24 h of incubation at 4 degrees C in the presence or absence of soluble asialofetuin (ASF). The asialoglycoprotein receptors were then immunolabeled by a rabbit antiserum followed by protein A-gold complexes. Average labeling was 41 +/- 2 particles x microns -2 (mean +/- SEM, n = 50), corresponding to an efficiency of about 25% as compared to radioligand binding data. Nearest neighbor analysis of gold particles showed an almost random distribution on cells incubated without asialofetuin, but significant clustering after exposure to the ligand. In a second series of experiments, hepatocytes were incubated at 4 degrees C for 2 or 24 h with asialofetuin adsorbed onto 15-nm gold particles (2 x 10(12) particles x ml-1, 32 molecules x particle-1), then fixed. On average, 20 +/- 2 (n = 14) and 38 +/- 6 (n = 19) particles x microns -2 were observed after 2 and 24 h, respectively. By comparison to control preparations, clustering of particles was found at both time intervals, although no significant difference could be detected between 2 and 24 h using the Kolmogorov-Smirnov test when the abundance of particles was taken into consideration. Summarizing, (1) in absence of ligand, the asialoglycoprotein receptor is almost randomly distributed on rat hepatocytes, (2) both soluble ASF and ASF-gold complexes induce receptor clustering, (3) even at 4 degrees C, the plasma membrane remains sufficiently fluid for receptor clustering.