Structure-function dissection of D6, an atypical scavenger receptor.
Chemokines direct leukocyte migration by activating intracellular signalling pathways through G-protein coupled chemokine receptors. However, they also bind to other surface proteins, including a group of molecules which we refer to as 'atypical' chemokine receptors. One such molecule is D6. D6 is structurally-related to other chemokine receptors, and binds specific pro-inflammatory chemokines with high affinity, but surprisingly, when expressed in heterologous cell lines, it is unable to transduce signals after chemokine engagement. Instead, by using the approaches outlined in this chapter, evidence has emerged that D6 acts as a chemokine scavenger which uses unique intracellular trafficking properties to continuously sequester extracellular chemokines into cells. It is envisaged that this suppresses inflammation in vivo by limiting pro-inflammatory chemokine bioavailability, and indeed, D6 deficient mice show exaggerated inflammatory responses to a variety of challenges. In addition to the in vitro functional studies, we also describe the methods we have used to express, purify and analyse large quantities of D6 protein. The unusually high stability of D6 and its broad subcellular distribution enables D6 to be expressed to very high levels in transfected cells, making it possible, at least in principal, to produce enough D6 to allow for purification of quantities suitable for crystallisation. This is a key step on the path towards generating a three-dimensional structure of the molecule. Thus, the protocols we outline have helped establish chemokine scavenging as a novel paradigm in chemokine biology, and may also ultimately provide unprecedented insight into the structure of D6 and other chemokine receptors.
Nibbs
Robert J B
RJ
Division of Immunology, Infection and Inflammation, Glasgow Biomedical Research Center, Glasgow University, Glasgow, United Kingdom.
McLean
Pauline
P
McCulloch
Clare
C
Riboldi-Tunnicliffe
Alan
A
Blair
Emma
E
Zhu
Yanshi
Y
Isaacs
Neil
N
Graham
Gerard J
GJ
D000818:Animals; D015153:Blotting, Western; D005434:Flow Cytometry; D006801:Humans; D051379:Mice; D054447:Receptors, CCR10 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000097628:Chemokine Receptor D6
D054447:Receptors, CCR10; D000097628:Chemokine Receptor D6