Analysis of Chemokine Receptor Trafficking by Site-Specific Biotinylation

Abstract

Chemokine receptors undergo internalization and desensitization in response to ligand activation. Internalized receptors are either preferentially directed towards recycling pathways (e.g. CCR5) or sorted for proteasomal degradation (e.g. CXCR4). Here we describe a method for the analysis of receptor internalization and recycling based on specific Bir A-mediated biotinylation of an acceptor peptide coupled to the receptor, which allows a more detailed analysis of receptor trafficking compared to classical antibody-based detection methods. Studies on constitutive internalization of the chemokine receptors CXCR4 (12.1% ± 0.99% receptor internalization/h) and CCR5 (13.7% ± 0.68%/h) reveals modulation of these processes by inverse (TAK779; 10.9% ± 0.95%/h) or partial agonists (Met-CCL5; 15.6% ± 0.5%/h). These results suggest an actively driven internalization process. We also demonstrate the advantages of specific biotinylation compared to classical antibody detection during agonist-induced receptor internalization, which may be used for immunofluorescence analysis as well. Site-specific biotinylation may be applicable to studies on trafficking of transmembrane proteins, in general.

CXCR4 expression in tumor associated cells in blood is prognostic for progression and survival in pancreatic cancer

“…It was observed that after exposure to the isoproterenol agonist, both internalization and recycling of the CXCR4 could be seen. Interestingly, this internalization and subsequent recycling of CXCR4 receptor has been demonstrated in other solid cancer types [ 23 ]. The highest level of CXCR4 expression occurred at one-hour post exposure to isoproterenol at 20 μM which has been observed in previous works on the stimulation of cancer cells with beta agonists [ 22 , 23 ].…”

Biochemical regulation and structural analysis of copper‐transporting ATPase in a human hepatoma cell line for Wilson disease

“…Thus, the structural dynamics of mutated 2EW9 and modeled ATPase defines the effect of single nucleotide mutation, in one of the known missense mutations causing WD in the ATP7B, ATP7B9, and ATP7BL region of the protein . Furthermore, RMSD, RMSF, and root mean gyration analyses provide substantial support to the results of wet lab observation that ATP7BL mutation has maximum fluctuations and thus, causing more intake of copper binding resulting into significant damaging effect . In summary, the mutated structures of ATPase taken along with the prior solved structures of the other ATPase enzymes explains the effect as damaging or disruptive of a WD causing mutation present in the proteins regulatory region, where very few disease‐causing mutations have been reported.…”

Evolution of chemokine receptors is driven by mutations in the sodium binding site

“…A unique Kd in the nanomolar range is observed for CXCL12 binding to CXCR4 [ 44 ]; (3) Constitutive activity: CCR5 is partially constitutively active for both the Gαi pathway and the β-arrestin mediated internalization [ 45 , 46 ]. Wild type CXCR4 undergoes constitutive internalization by a β-arrestin pathway [ 47 , 48 ] but constitutive G protein activity of CXCR4 has been observed only upon mutation of N3.35 to Ser or Ala [ 23 ]. In spite of these differences, it is interesting to note that, as observed for other chemokine receptors, CXCR4 and CCR5 do not possess an ionic lock [ 42 ] between position 3.50 (Arg in both receptors) and position 6.30 (Lys and Arg in CXCR4 and CCR5, respectively).…”