First identification of a chemotactic receptor in an invertebrate species: structural and functional characterization of Ciona intestinalis C3a receptor.
In mammals, the bioactive fragment C3a, released from C3 during complement activation, is a potent mediator of inflammatory reactions and exerts its functional activity through the specific binding to cell surface G protein-coupled seven-transmembrane receptors. Recently, we demonstrated a Ciona intestinalis C3a (CiC3a)-mediated chemotaxis of hemocytes in the deuterostome invertebrate Ciona intestinalis and suggested an important role for this molecule in inflammatory processes. In the present work, we have cloned and characterized the receptor molecule involved in the CiC3a-mediated chemotaxis and studied its expression profile. The sequence, encoding a 95,394 Da seven-transmembrane domain protein, shows the highest sequence homology with mammalian C3aRs. Northern blot analysis revealed that the CiC3aR is expressed abundantly in the heart and neural complex and to a lesser extent in the ovaries, hemocytes, and larvae. Three polyclonal Abs raised in rabbits against peptides corresponding to CiC3aR regions of the first and second extracellular loop and of the third intracellular loop react specifically in Western blotting with a single band of 98-102 kDa in hemocyte protein extracts. Immunostaining performed on circulating hemocytes with the three specific Abs revealed that CiC3aR is constitutively expressed only in hyaline and granular amoebocytes. In chemotaxis experiments, the Abs against the first and second extracellular loop inhibited directional migration of hemocytes toward the synthetic peptide reproducing the CiC3a C-terminal sequence, thus providing the compelling evidence that C. intestinalis expresses a functional C3aR homologous to the mammalian receptor. These findings further elucidate the evolutionary origin of the vertebrate complement-mediated proinflammatory process.
Melillo
Daniela
D
Laboratory of Cell Biology, Stazione Zoologica Anton Dohrn, Napoli, Italy.
Sfyroera
Georgia
G
De Santis
Rosaria
R
Graziano
Rita
R
Marino
Rita
R
Lambris
John D
JD
Pinto
Maria Rosaria
MR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002633:Chemotaxis / Q000276:immunology*; D002938:Ciona intestinalis / Q000276:immunology* / Q000378:metabolism; D015926:Complement C3a / Q000378:metabolism*; D020869:Gene Expression Profiling; D006434:Hemocytes / Q000737:chemistry / Q000502:physiology; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011951:Receptors, Complement / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013329:Structure-Activity Relationship
D008565:Membrane Proteins; D011951:Receptors, Complement; C085567:complement C3a receptor; D015926:Complement C3a