Geneeskundige Stichting Koningin Elisabeth ... - GSKE - FMRE

4. Purinergic receptors.
Purinergic receptors constitute a subfamily of G protein-coupled receptors that contains 5
functional receptors (P2Y 1, P2Y 2, P2Y 4, P2Y 6 and P2Y 11), and three of these receptors have
been described in our laboratory (P2Y 4, P2Y 6 and P2Y 11). During the last year, we have examined
the expression of mRNA for several P2Y nucleotide receptors by northern blot analysis in
purified type 1 cerebellar astrocyte cultures. These results suggest that different P2Y subtypes
could be responsible for ATP metabotropic calcium responses in these cells. To identify these
subtypes we have studied the pharmacological profile of ATP calcium responses using fura-2
microfluorimetry. All tested astrocytes responded to ATP and UTP stimulations evoking similar
calcium transients. Most astrocytes also responded to 2-methylthioATP and ADP challenges. The
agonist potency order was 2-methylthioATP > ADP > ATP = UTP. Cross-desensitization experiments
carried out with ATP, UTP, and 2-methylthioATP showed that 2-methylthioATP and UTP
interact with different receptors, P2Y 1 and P2Y 2 or P2Y 4. In a subpopulation of type 1 astrocytes,
ATP prestimulation did not block UTP responses, and UDP elicited clear intracellular Ca 2+
concentration responses at very low concentrations. 2-MethylthioATP and UTP calcium responses
exhibited different sensitivity to pertussis toxin and different inhibition patterns in response
to P2 antagonists. The P2Y 1-specific antagonist N:(6)-methyl-2'-deoxyadenosine 3', 5'-bisphosphate
(MRS 2179) specifically blocked the 2-methylthio-ATP responses. We can conclude that
all single astrocytes coexpressed at least two types of P2Y metabotropic receptors: P2Y 1 and
either P2Y 2 or P2Y 4 receptors. Moreover, 30-40% of astrocytes also coexpressed specific pyrimidine
receptors (Jimenez et al. 2000).
5. Chemokine receptors.
Chemokines are a superfamily of small signaling proteins that play major roles in the recruitment
of white blood cells toward the sites of inflammation. They play fundamental roles in the
physiology of acute and chronic inflammatory processes as well as in the pathological dysregulations
of these processes.
We have also continued to analyze the structure-function relationships of CCR5 and its chemokine
ligands. The reported structures of many CC chemokines show a conserved dimer interface
along their N-terminal region, raising the possibility that their quaternary arrangement
might influence their function. We have analyzed mutants of MIP-1β (generated by Patty
LiWang, College Station, Texas, USA) having a range of dimer K d values in order to determine
the significance of dimerization in receptor binding and cellular activation. Functional relevance
was determined by receptor binding affinity and the ability to invoke intracellular calcium
release from CHO cells transfected with the MIP-1β receptor CCR5. The monomeric N-terminally
truncated mutant MIP(9) was able to bind the CCR5 receptor with a K i of 600 pM but
displayed weak agonistic properties, while the monomeric mutant P8A still retained the ability
to tightly bind (K i = 480 pM) and to activate (EC 50 = 12 nM) the receptor. These data suggest
that the MIP-1β dimer is not required for CCR5 binding or activation. In addition, we identified
Phe13, the residue immediately following the conserved CC motif in MIP-1β, as a key determinant
for binding to CCR5. Replacement of Phe13 by Tyr, Leu, Lys, and Ala showed the aromatic
side chain to be important for both binding to CCR5 and chemokine dimerization (Laurence et
al. 2000).
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