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Pharmacological characterisation of dog recombinant CRTh2 In humans, CRTh2 is expressed on Th2 lymphocytes, eosinophils and basophils. The natural CRTh2 agonists, prostaglandin D2 (PGD2) and some of its metabolites, promote a number of responses including Ca2+ flux, CD11b up-regulation, shape change and chemotaxis (Hirai et al., 2001). These responses have not been reproduced in isolated rodent cells making interpretation of the role of CRTh2 in these species difficult. In dogs, PGD2 has been reported to cause eosinophil release in perfused trachea (Emery et al., 1989) but it is not known whether this response involves CRTh2. Understanding of the role of CRTh2 in this response relies on well-characterised ligands for dog CRTh2. The aim of this study was to clone and express dog CRTh2 and to compare binding of a range of ligands with previously described recombinant CRTh2 from human, rat, mouse and guinea pig. A DNA fragment encoding a portion of dog CRTh2 was cloned using consensus PCR primers designed from an alignment of published CRTh2 sequences. The complete coding sequence was obtained using a combination of inverse PCR of dog genomic DNA and public domain data (NCBI trace archive). Recombinant CRTh2 was expressed in HEK 293 cells. Receptor binding studies used isolated membranes in a scintillation proximity assay. All data are expressed as mean ± SEM (or SD when n=2). Dog CRTh2 had 84% identity to human CRTh2 and ~70% identity to mouse, rat and guinea pig CRTh2 at the protein level. When expressed in HEK 293 cells, dog CRTh2 bound 3H-PGD2 with a Kd of 6.5±1.7 nM (n=3), which was similar to the affinity of 3H-PGD2 for human (5.1±1.2 nM n=4), rat (5.5±0.4 nM n=2), mouse (7.9±3.6 nM n=2) and guinea pig (6.0±5.0 nM, n=2) CRTh2. Known CRTh2 ligands displaced 3H-PGD 2 from CRTh2 of dog and other species (Table 1). In contrast, a range of agonists and antagonists at related receptors (PGE 2, LTB 4, U46619, BW245C and BW868C) all poorly displaced 3H-PGD2 binding (pIC50’s <6.0, n=4). Table 1 pIC50 for displacement of 3H-PGD2 from CRTh2 (n = 4-14).
Our results show that dog CRTh2 is closely related to CRTh2 from human, rat, mouse and guinea pig. Furthermore, a range of ligands bind to recombinant dog CRTh2 with similar affinity to recombinant CRTh2 from the other four species. This highlights that DK-PGD 2 is an appropriate tool to probe the functional response of CRTh2 in the dog. Marshall et al., this meeting).
Emery, DL et al., (1989) J. Appl. Physiol .67:959-962. |
