Abstract
The mechanisms that regulate the transport of the macrophage class A scavenger receptor during ligand uptake were investigated. Kinetic analysis of the changes in receptor phosphorylation demonstrated that serine phosphorylation increased during the internalization of acetyl-low density lipoproteins (LDL) by macrophages. The increase was maximal at about 2.5 min after the initiation of ligand uptake. Oxidized LDL also stimulated serine phosphorylation, but the relative increase was smaller and the time to maximum was shorter. Receptor mutants expressed in Chinese hamster ovary and COS cells showed that elimination of the potential phosphorylation site at Ser(21) increased acetyl-LDL metabolism, whereas inactivation of the site at Ser(49) reduced acetyl-LDL uptake. The increase in uptake by the Ser(21) mutant was due to an increase in surface receptor expression. In contrast, elimination of the site at Ser(49) did not affect receptor expression but slowed receptor internalization. To identify potential internalization signal sequences, beta-turn structure in the cytosolic domain was targeted for mutagenesis. Disruption of one region near Asp(25) inhibited receptor activity. The studies support a model whereby receptor internalization requires the presence of an internalization signal motif but that the rate of receptor internalization is governed by the pattern of receptor phosphorylation induced by the ligand.
Highlights
We have shown previously that the mouse macrophage scavenger receptor is phosphorylated in situ [22], but the relation of this to receptor transport has not yet been established
Cells were incubated with acetyl-low density lipoprotein (LDL) at 4 °C to allow surface binding and washed to remove non-bound lipoprotein
The internalization of ligands by the scavenger receptor is thought to follow classical coated pit-dependent endocytosis. This is based on ultrastructural studies that have shown that after ligand binding, there is a rapid association of receptorligand complexes with coated pit structures (15, 16, 19 –21)
Summary
Materials—Carrier-free Na125I and [14C]chloramphenicol were purchased from Amersham Pharmacia Biotech. The scavenger receptor DNA preparations (7.5 g) were diluted in Tris-EDTA buffer (20 l) and added to 15-ml culture tubes To this was added 6 ml of a freshly prepared stock solution containing 400 g/ml DEAE-dextran, 0.1 mM chloroquine, and 1.25 g/ml of the reporter gene construct pcDNA/CAT in DMEM, 10% NS. These were incubated at room temperature for 15 min and added to the washed cells. Statistics—Statistical analysis was done using non-paired Student’s t test
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