Functional roles of short sequence motifs in the endocytosis of membrane receptors

Abstract

Internalization and trafficking of cell-surface membrane receptors and proteins into subcellular compartments is mediated by specific short-sequence signal motifs, which are usually located within the cytoplasmic domains of these receptor and protein molecules. The signals usually consist of short linear amino acid sequences, which are recognized by adaptor coat proteins along the endocytic and sorting pathways. The complex arrays of signals and recognition proteins ensure the dynamic movement, accurate trafficking, and designated distribution of transmembrane receptors and ligands into intracellular compartments, particularly to the endosomal-lysosomal system. This review summarizes the new information and concepts, integrating them with the current and established views of endocytosis, intracellular trafficking, and sorting of membrane receptors and proteins. Particular emphasis has been given to the functional roles of short-sequence signal motifs responsible for the itinerary and destination of membrane receptors and proteins moving into the subcellular compartments. The specific characteristics and functions of short-sequence motifs, including various tyrosine-based, dileucine-type, and other short-sequence signals in the trafficking and sorting of membrane receptors and membrane proteins are presented and discussed.

Figure 1

Diagrammatic representation of intracellular pathways of receptor-mediated endocytosis and trafficking: The ligand binding to specific cell surface receptor leads to a selective recruitment of ligand-receptor complexes into clathrin-coated pits. The coated pit represents a small area of the plasma membrane, which invaginates and pinch-off into vesicle in the cytosol. Coated pits and vesicles also trigger the recruitment of adapter proteins for example AP-2 and other interacting protein molecules. The caveolin pathway also internalizes the cargo complex, independent of clathrin-coated pits and the surface of caveolae is coated by caveolin. Both clathrin- and caveolin-dependent routes require dynamin protein to achieve the fission of the membrane invaginations and vesicle internalization. The ligand-receptor complexes within the cargo entering via the clathrin and caveolin pathways are usually directed to early endosomes. From the endosomes, the receptors and ligands are sorted to various subcellular locations, where the internalized molecules are either routed to degradative compartments such as the late endosomes, and/or lysosomes, or recycled to the plasma membrane via recycling endosomes. The recycled molecules can participate in several rounds of endocytosis. Alternatively, the internalized cargo molecules may be sequestered in endosomes for a longer period of time and continue to spark signaling events. Some early and late endosomes also contain membrane structures in the lumen, which are referred to as multi-vesicular bodies (MVBs). The endosome and lysosome system can also transmit and receive cargo from trans-Golgi network (TGN) involving vesicular intermediate carriers. The key proteins involved in the trafficking of molecules at different locations have been indicated such as AP-1, AP-1A, AP-1B, AP-2, AP-4, Dab-1, Esp15, GGA, PACS-1, and TIP.

Figure 2

Requirements of GDAY sequence for Internalization of guanylyl cyclase/natriuretic peptide in HEK-293 Cells: Mutagenesis of Gly-Asp-Ala-Tyr (GDAY) sequence of guanylyl cyclase/natriuretic peptide receptor-A (GCA/NPRA) and expression of both wild-type and GDAY/AAAA mutant receptors in human embryonic kidney-293 (HEK-293) cells. (A) Alanine substitutions at amino acid residues 918–925. (B) Alanine substitutions indicating two residues in different combinations in GDAY motif. Confluent HEK-293 cells expressing either wild-type or mutant receptors were washed twice with 2 ml of assay medium (Dulbecco’s modified Eagle’s medium containing 0.1% bovine serum albumin) and then exposed to ¹²⁵I-ANP at 4˚C for 1 h in the absence or presence of unlabeled ANP. After which, the cells were washed four times with assay medium and reincubated in 2 ml of fresh medium at 37˚C. After 10 min internalization and incubation period, the internalization of ligand-receptor complexes was quantified. Figure has been reproduced from the previous publication and has been adapted with the permission (57).

Figure 3

Competition binding of ¹²⁵I-ANP in HEK-293 cells expressing wild-type or GDAY/AAAA mutant receptors: Confluent HEK-293 cells were cultured in 6 cm² dishes and incubated in 2 ml of assay medium with 1 nM ¹²⁵I-ANP and increasing concentrations of unlabelled ANP at 4 °C for 1 h. Cells were washed four times, each with 2 ml of assay medium to remove the unbound radioligand, and then dissolved in 1 M NaOH. Specific ¹²⁵I-ANP radioactivity was determined in the solubilized cell extract and the binding curves are derived from the specific binding data. The non-specific binding was determined by using 100-fold excess molar concentrations of unlabelled ANP. The Kd values and receptor densities (Bmax) were determined from the binding competition and by Scatchard analysis of the ¹²⁵I-ANP binding data. Figure has been reproduced from the previous publication and has been adapted with the permission (57).

Figure 4

Schematic representation of internalization, recycling and intracellular degradation of membrane receptors in intact cells: The schematic diagram shown postulates the stoichimetric kinetics of internalization, subcellular sequestration, recycling, and ultimately metabolic turnover of ligand-receptor complexes from cell surface to cell interior and back to the plasma membrane. The scheme depicts that after synthesis: a) the receptor is inserted in the plasma membrane, b) the ligand-receptor complex enters the cell via coated pits, and c) the complex is processed intracellularly through endosome, lysosome, and/or chloroquine-insensitive pathways. Sorting of bound ligand-receptor complexes into the intracellular compartments may occur by i) lysosomal degradative metabolic pathway, ii) endosomal dissociation metabolic pathway, and/or iii) release through the chloroquine-insensitive pathway. The cardiac hormone atrial natriuretic peptide (ANP) and its biologically active membrane receptor have been exemplified. Figure has been reproduced from the previous publication and has been adapted with the permission (13).