Cross-desensitization of chemoattractant receptors occurs at multiple levels. Evidence for a role for inhibition of phospholipase C activity.
To define the molecular mechanisms of cross-regulation among chemoattractant receptors, we stably coexpressed, in a rat basophilic leukemia (RBL-2H3) cell line, epitope-tagged receptors for the chemoattractants formylmethionylleucylphenylalanine (fMLP), a peptide of the fifth component of the complement system (C5a), and interleukin-8 (IL-8). All the expressed receptors underwent homologous phosphorylation and desensitization upon agonist stimulation. When co-expressed, epitope-tagged C5a receptor (ET-C5aR) and epitope-tagged IL-8 receptor (ET-IL-8RA) were cross-phosphorylated by activation of the other. Activation of epitope-tagged fMLP receptor (ET-FR) also cross-phosphorylated ET-C5aR and ET-IL-8RA, but ET-FR was totally resistant to cross-phosphorylation. Similarly, C5a and IL-8 stimulation of [35S]guanosine 5'-3-O-(thio) triphosphate (GTP gamma S) binding and Ca2+ mobilization were cross-desensitized by each other and by fMLP. Stimulation of [35S]GTP gamma S binding by fMLP was also not cross-desensitized by C5a or IL-8, however, Ca2+ mobilization was, suggesting a site of inhibition distal to G protein activation. Consistent with this desensitization of Ca2+ mobilization, inositol 1,4,5-trisphosphate release in RBL-2H3 cells expressing both ET-C5aR and ET-FR revealed that fMLP and C5a cross-desensitized each other's ability to stimulate phosphoinositide hydrolysis. Taken together, these results indicate that receptor cross-phosphorylation correlates directly with desensitization at the level of G protein activation. The ET-FR was resistant to this process. Of note, cross-desensitization of ET-FR at the level of phosphoinositide hydrolysis and Ca2+ mobilization was demonstrated in the absence of receptor phosphorylation. This suggests a new form of chemoattractant cross-regulation at a site distal to receptor/G protein coupling, involving the activity of phospholipase C.
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D015936:Complement C5a / Q000494:pharmacology*; D000939:Epitopes; D015870:Gene Expression; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism; D016209:Interleukin-8 / Q000494:pharmacology*; D015471:Leukemia, Basophilic, Acute; D008969:Molecular Sequence Data; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D010766:Phosphorylation; D051381:Rats; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000096:biosynthesis / Q000502:physiology*; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000096:biosynthesis / Q000502:physiology*; D018123:Receptors, Interleukin / Q000096:biosynthesis / Q000502:physiology*; D023062:Receptors, Interleukin-8A; D018000:Receptors, Peptide / Q000096:biosynthesis / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D015703:Antigens, CD; D000939:Epitopes; D007295:Inositol Phosphates; D016209:Interleukin-8; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018123:Receptors, Interleukin; D023062:Receptors, Interleukin-8A; D018000:Receptors, Peptide; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009240:N-Formylmethionine Leucyl-Phenylalanine; D015936:Complement C5a; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases; D002118:Calcium