A comparison of post-receptor signal transduction events in Jurkat cells transfected with either IL-8R1 or IL-8R2. Chemokine mediated activation of p42/p44 MAP-kinase (ERK-2).
The CXC chemokine, IL-8, is a potent chemoattractant of neutrophils and binds to two distinct receptors, termed IL-8R1 and IL-8R2. These receptors share high affinity for IL-8, however, only IL-8R1 is specific for IL-8 whereas IL-8R2 binds other related chemokines, including GRO alpha with high affinity. Stable Jurkat transfectants were generated expressing either functional IL-8R1 or IL-8R2 (J-IL8R1 and J-IL8R2). Both J-IL8R1 and J-IL8R2 exhibited high affinity IL-8 binding (Kd 3-5 nM) with respective receptor densities of 23,000 +/- 3,000 and 18,500 +/- 1,500. Pre-treatment of both transfectants with 1.0 micrograms/ml B. pertussis toxin (PTx) resulted in inhibition of IL-8 mediated intracellular Ca2+ mobilisation and chemotaxis, without altering the receptor's affinity for its ligand. This indicates that both receptors couple to a PTx-sensitive G-protein. Further studies showed that IL-8R1 and IL-8R2 could mediate time-dependent phosphorylation of p42/p44 MAP-kinase. In both transfectants, phosphorylation was maximal at 1-2 min after IL-8 stimulation and could be inhibited by PTx. Stimulation of J-IL8R1 and J-IL8R2 with GRO alpha revealed that this chemokine was a more potent activator of MAP-kinase in J-IL8R2, an observation reflected in the high affinity binding of GRO alpha to IL-8R2. These studies indicate that chemokines are capable of activating protein kinases and with regards to PTx-sensitivity and MAP-kinase stimulation, no significant differences between IL-8R1 and IL-8R2 post-receptor signalling occur during cell activation by IL-8.
Jones
S A
SA
Theodor-Kocher Institute, University of Bern, Switzerland.
Moser
B
B
Thelen
M
M
D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D016209:Interleukin-8 / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1; D037342:Pertussis Toxin; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D018123:Receptors, Interleukin / Q000235:genetics / Q000378:metabolism*; D023062:Receptors, Interleukin-8A; D015398:Signal Transduction; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D016209:Interleukin-8; D018123:Receptors, Interleukin; D023062:Receptors, Interleukin-8A; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019950:Mitogen-Activated Protein Kinase 1; D019204:GTP-Binding Proteins