Expression of a Functional Anaphylatoxin C3a Receptor by Astrocytes

Abstract: Human astrocyte cell lines reportedly contain a specific receptor for the complement anaphylatoxin C3a based on ligand-binding studies, functional responses, and RNA analysis by RT-PCR. Uptake of ¹²⁵I-C3a by astrocytes was specific and reversible. Scatchard analysis indicated the presence of two classes of binding sites. High-affinity binding sites were abundantly expressed (20,000–80,000 sites per cell) with an estimated K _D of 1–2 n M . Low-affinity binding sites with a K _D of 209 n M were largely expressed ( n ≥ 4 × 10⁶ sites per cell) and probably did not reflect a receptor-mediated binding, but rather an ionic interaction between C3a and the membrane. Analysis of astrocyte mRNA by RT-PCR with three different sets of primers covering 60% of the C3a receptor (C3aR) mRNA sequence indicated that glial C3aR was identical to the leukocytic one. Western blot analysis using a specific anti-C3aR evidenced a C3aR with a molecular mass of 60,000 Da. C3a and a superagonist peptide, E7, induced a transient increase of intracellular [Ca²⁺] in primary culture of astrocytes. Treatment of the ligands by carboxypeptidase B to eliminate the C-terminus Arg considerably decreased the [Ca²⁺] response. Moreover, flow cytometry experiments demonstrated the expression of C3aR on normal rat astrocyte membrane. This report brings new insight for the role of the complement system in the brain inflammation response.