Functional reconstitution of a receptor-activated signal transduction pathway in Xenopus laevis oocytes using the cloned human C5a receptor.
We have used the polymerase chain reaction to isolate and clone the cDNA encoding the human C5a receptor, and have injected the cDNA-derived receptor cRNA into Xenopus laevis oocytes for functional characterization of the receptor protein. Receptor activity was determined either electrophysiologically by measuring the agonist-dependent opening of [Ca2+]i-dependent Cl- channels, or by analysing the agonist-dependent efflux of 45Ca2+ from the oocytes. Using both methodologies, injection of pure C5a receptor cRNA failed to confer C5a sensitivity on the oocytes. In contrast, marked responses to C5a were observed when the receptor cRNA was supplemented with poly(A)+ RNA isolated from undifferentiated HL-60 cells, which is devoid of C5a receptor mRNA. Binding studies using radioiodinated C5a revealed that the C5a receptor polypeptide was in fact synthesized and targeted to the oocyte plasma membrane in oocytes injected with receptor cRNA alone, and that the level of receptor expression was not influenced by coinjection of poly(A)+ RNA from undifferentiated HL-60 cells. These results strongly suggest that the human C5a receptor requires a specific cofactor(s) lacking in Xenopus oocytes but present in undifferentiated HL-60 cells, to generate intracellular signals in oocytes. Identification and characterization of this factor will provide important information about the molecular mechanisms by which G-protein-coupled receptors activate phospholipase C.
Schultz
P
P
Pharmakologisches Institut, Universität Heidelberg, Germany.
Stannek
P
P
Bischoff
S C
SC
Dahinden
C A
CA
Gierschik
P
P
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D015936:Complement C5a / Q000235:genetics / Q000502:physiology*; D005260:Female; D005816:Genetic Complementation Test; D006801:Humans; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism / Q000502:physiology*; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D014982:Xenopus laevis
D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D015936:Complement C5a