CXCR4 receptor phosphorylation

Arrestin recruitment can also be compared between diiierent substitution point mutants of the same receptor. Such comparisons may yield information about key residues for arrestin recruitment, for example, the respective roles of specific phosphorylation sites in the receptor C-terminal (BusiUo et al., 2010 de Mimnik et al., 2015). Substitution of some of these potential phosphorylation sites, but not others, may result in the absence of detectable arrestin recruitment. One conundrum of such analyses is that amino acid substitutions in the receptor may alter the sensitivity of the BRET system via conformational effects. This is illustrated by an example in which BusiUo et al. foimd that alanine substitutions of phosphorylation sites in the CXCR4 C-terminal could lead to increased BB T efficiency—the authors concluded a conformational effect (BusiUo et al., 2010). Because these effects may also decrease (or abolish) BRET efficiency for purely technical reasons (and not as an intrinsic property of the mutant receptors), we recommend to gather complementary evidence before concluding on the efficacy of arrestin recruitment. Alternative interpretations such as the occurrence of different arrestin recruitment modalities to different receptor mutants should also be considered. This applies in particular to mutations of phosphorylation sites, as the intricate receptor phosphorylation patterns (bar-codes) by multiple kinases are known to determine the recruited arrestin species (Nobles et al., 2011). 

Upon activation by CXCLl2, CXCR4 is quickly phosphorylated and internalized. Several residues in its C-terminus tail have been identified as potential phosphorylation sites by truncation and mutagenesis studies as reviewed in Busillo and Benovic (2007). Removal of 45 amino acid residues from the C-terminal of CXCR4 led to elimination of CXCL12-induced phoshorylation, enhanced receptor activity... 

Majka, M., Ratajczak, J., et al. (2000). Binding of stromal derived factor-lalpha (SDF-lalpha) to CXCR4 chemokine receptor in normal human megakaryoblasts but not in platelets induces phosphorylation of mitogen-activated protein kinase p42/44 (MAPK), ELK-1 transcription factor and serine/threonine kinase AKT. Eur J Haematol 64(3) 164—72. 

I., Sozzani, S., Peiper, S.C., Horuk, R., Ali, H., and Snyderman, R. (1997) Regulation of human chemokine receptors CXCR4. Role of phosphorylation in desensitization and internalization. The Journal of Biological Chemistry, 272, 28726-28731. 

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