Abstract
Cloning of rat prolactin receptor (PRLR) cDNAs revealed the existence of two isoforms, termed short and long according to the length of their cytoplasmic domain. Internalization studies show, first, that PRLR internalization is hormone-dependent and, second, that ligand-receptor complexes of the short PRLR are internalized to a larger extent compared to the long form. In order to identify regions within the cytoplasmic domain of the short PRLR required for efficient internalization, serial truncations of the cytoplasmic tail were performed by inserting a stop codon in place of those encoding residues 282, 273, 262, 253, 244, or 237 (wild type short PRLR contains 291 amino acids). Our data show that two motifs, lying within residues 253-261 and 273-281, are involved in internalization. Both regions contain a consensus feature identified within other receptors as internalization signals, namely a di-leucine peptide (amino acids 259-260) and a tetrapeptide predicted to adopt a beta-turn structure (amino acids 276-279). We propose these two motifs are involved in PRLR endocytosis. Finally, we show that alpha-adaptin, a component of adaptor protein AP-2, coprecipitates with short PRLR complexes upon PRL stimulation, which strongly suggests that PRLR internalization is mediated by the clathrin-coated pits endocytotic pathway.
Highlights
Receptor-mediated endocytosis is the mechanism by which a variety of nutrients, hormones, and growth factors are and efficiently transported into the cell
Specific binding to cell surface receptors after 6 h of incubation at 4 °C was similar for both forms of receptor (ϳ3–5% of total radioactivity), it clearly appears that the short form complexed with the ligand is internalized to a greater extent than the long form (80% versus 50% of internalization after 120 min)
The total number of binding sites observed in cells expressing T236 prolactin receptor (PRLR) is 3 times higher compared to the wild type receptor
Summary
Receptor-mediated endocytosis is the mechanism by which a variety of nutrients, hormones, and growth factors are and efficiently transported into the cell During this process, receptors are selectively concentrated in clathrin-coated pits from which they are rapidly internalized and delivered to endosomes (for review, see Ref. 1). Endocytotic codes have been identified as short linear arrays of amino acids which, despite their low degree of sequence similarity, are all assumed to fold in a tight -turn [6, 7] These motifs may be recognized by plasma membrane-associated adaptor proteins, or AP-2, which mediate the recruitment of these receptors into clathrin-coated pits and promote high-efficiency endocytosis Our study demonstrates that two regions, each containing consensus features previously reported to mediate receptor internalization, are involved in PRLR endocytosis
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