The human chemokine receptor CXCR4 is a member of the G-protein coupled receptor (GPCR) superfamily of proteins, which comprise approximately 30% of current drug targets. Activation of CXCR4 by its endogenous ligand CXCL12 (SDF-1a), is critical for development and cell migration. The upregulation of CXCR4 in a variety of human cancers, and its role as an HIV co-receptor makes CXCR4 an increasingly important therapeutic target. Here we report a comprehensive functional analysis of CXCR4, identifying residues required for CXCL12-mediated signal transduction, complementing the recently published crystal structure of CXCR4. Using Shotgun Mutagenesis technology, we created a complete mutation library, individually targeting each of the 352 CXCR4 residues. Each CXCR4 variant was tested for protein expression, conformation, and function. Using this approach, we identified critical residues for CXCL12-mediated CXCR4 signal transduction, largely occupying the receptor transmembrane domains. The majority of critical residues do not appear to be involved in ligand binding or G-protein coupling, but rather cluster within specific alpha-helices that are known in GPCR family receptors to undergo significant conformational changes upon activation. Our results are consistent with a conserved structure-based hypothesis of GPCR signal transduction and suggest that the critical residues identified here are those that are required to mediate CXCR4 receptor signaling. These data may facilitate the elucidation of mechanisms underlying the transduction of GPCR signaling in general and will also aid in drug design, providing insight for the specific targeting of residues that do not disrupt normal CXCL12-mediated CXCR4 signaling.

Structural mechanism by which the GPCR CXCR4 transduces signaling events

244th National Meeting (2012)

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