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Releasable SNAP-tag probes for studying endocytosis and recycling.

ABSTRACT: Site-specific labeling of cellular proteins with chemical probes is a powerful tool for live cell imaging of biological processes. One popular system, known as the SNAP-tag, is based on an engineered variant of the 20-kDa DNA repair protein O(6)-alkylguanine-DNA-alkyltransferase (AGT) that covalently reacts with O(6)-benzylguanine (BG) and can be derivatized with a number of reporter groups. For studying the endocytosis and recycling of cell surface proteins, the covalent nature of BG binding to the SNAP-tag is problematic, since removing excess noninternalized probe from the cell surface is not feasible. Here we describe a modification of the SNAP-tag technology that permits the rapid release of fluorescently labeled probes from the cell surface without affecting the population of labeled molecules sequestered within endosomes. This simple yet effective approach allows quantitative measurements of endocytosis and recycling in both imaging and biochemical assays and is especially useful when studying endosomal dynamics in live cells.

SUBMITTER: Cole NB

PROVIDER: S-EPMC3307366 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Releasable SNAP-tag probes for studying endocytosis and recycling.

Cole Nelson B NB Donaldson Julie G JG

ACS chemical biology 20120113 3

Site-specific labeling of cellular proteins with chemical probes is a powerful tool for live cell imaging of biological processes. One popular system, known as the SNAP-tag, is based on an engineered variant of the 20-kDa DNA repair protein O(6)-alkylguanine-DNA-alkyltransferase (AGT) that covalently reacts with O(6)-benzylguanine (BG) and can be derivatized with a number of reporter groups. For studying the endocytosis and recycling of cell surface proteins, the covalent nature of BG binding to ...[more]

PMID: 22216966

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Project description:The chemokine receptor CCR5 is a cofactor for the entry of R5 tropic strains of human immunodeficiency viruses (HIV)-1 and -2 and simian immunodeficiency virus. Cells susceptible to infection by these viruses can be protected by treatment with the CCR5 ligands regulated on activation, normal T cell expressed and secreted (RANTES), MIP-1alpha, and MIP-1beta. A major component of the mechanism through which chemokines protect cells from HIV infection is by inducing endocytosis of the chemokine receptor. Aminooxypentane (AOP)-RANTES, an NH(2)-terminal modified form of RANTES, is a potent inhibitor of infection by R5 HIV strains. AOP-RANTES efficiently downmodulates the cell surface expression of CCR5 and, in contrast with RANTES, appears to prevent recycling of CCR5 to the cell surface. Here, we investigate the cellular basis of this effect. Using CHO cells expressing human CCR5, we show that both RANTES and AOP-RANTES induce rapid internalization of CCR5. In the absence of ligand, CCR5 shows constitutive turnover with a half-time of 6-9 h. Addition of RANTES or AOP-RANTES has little effect on the rate of CCR5 turnover. Immunofluorescence and immunoelectron microscopy show that most of the CCR5 internalized after RANTES or AOP-RANTES treatment accumulates in small membrane-bound vesicles and tubules clustered in the perinuclear region of the cell. Colocalization with transferrin receptors in the same clusters of vesicles indicates that CCR5 accumulates in recycling endosomes. After the removal of RANTES, internalized CCR5 recycles to the cell surface and is sensitive to further rounds of RANTES-induced endocytosis. In contrast, after the removal of AOP-RANTES, most CCR5 remains intracellular. We show that these CCR5 molecules do recycle to the cell surface, with kinetics equivalent to those of receptors in RANTES-treated cells. However, these recycled CCR5 molecules are rapidly reinternalized. Our results indicate that AOP-RANTES-induced changes in CCR5 alter the steady-state distribution of the receptor and provide the first evidence for G protein-coupled receptor trafficking through the recycling endosome compartment.

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Releasable SNAP-tag probes for studying endocytosis and recycling.

Publications

Releasable SNAP-tag probes for studying endocytosis and recycling.

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