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Binding of leukotriene B4 and its analogs to human polymorphonuclear leukocyte membrane receptors
1Arthritis-Immunology Center, Veterans Administration Medical Center, Medical College of Pennsylvania, Philadelphia 19104.
Abstract:
LTB4-induced proinflammatory responses in PMN including chemotaxis, chemokinesis, aggregation and degranulation are thought to be initiated through the binding of LTB4 to membrane receptors. To explore further the nature of this binding, we have established a receptor binding assay to investigate the structural specificity requirements for agonist binding. Human PMN plasma membrane was enriched by homogenization and discontinuous sucrose density gradient purification. [3H]-LTB4 binding to the purified membrane was dependent on the concentration of membrane protein and the time of incubation. At 20 degrees C, binding of [3H]-LTB4 to the membrane receptor was rapid, required 8 to 10 min to reach a steady-state and remained stable for up to 50 min. Equilibrium saturation binding studies showed that [3H]-LTB4 bound to high affinity (dissociation constant, Kd = 1.5 nM), and low capacity (density, Bmax = 40 pmol/mg protein) receptor sites. Competition binding studies showed that LTB4, LTB4-epimers, 20-OH-LTB4, 2-nor-LTB4, 6-trans-epi-LTB4 and 6-trans-LTB4, in decreasing order of affinity, bound to the [3H]-LTB4 receptors. The mean binding affinities (Ki) of these analogs were 2, 34, 58, 80, 1075 and 1275 nM, respectively. Thus, optimal binding to the receptors requires stereospecific 5(S), 12(R) hydroxyl groups, a cis-double bond at C-6, and a full length eicosanoid backbone. The binding affinity and rank-order potency of these analogs correlated with their intrinsic agonistic activities in inducing PMN chemotaxis. These studies have demonstrated the existence of high affinity, stereoselective and specific receptors for LTB4 in human PMN plasma membrane.
Insights
This study identifies specific structural requirements for leukotriene B4 (LTB4) binding to its receptors on human neutrophils. These findings clarify how LTB4 triggers inflammatory responses.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Leukotriene B4 (LTB4) is a potent mediator of inflammation, inducing neutrophil (PMN) responses like chemotaxis and degranulation.
- These responses are initiated by LTB4 binding to specific membrane receptors on PMNs.
- Understanding the structural basis of this binding is crucial for comprehending LTB4's role in inflammation.
Purpose of the Study:
- To establish a receptor binding assay for human PMN plasma membrane.
- To investigate the structural specificity requirements for LTB4 agonist binding.
- To characterize the affinity and capacity of LTB4 receptors.
Main Methods:
- Human PMN plasma membranes were isolated using homogenization and sucrose density gradient purification.
- A radioligand binding assay using [3H]-LTB4 was developed to measure receptor binding.
- Competition binding studies were performed with various LTB4 analogs to determine binding affinities and structural requirements.
Main Results:
- [3H]-LTB4 binding was time- and concentration-dependent, reaching steady-state within 8-10 minutes at 20°C.
- The receptor exhibited high affinity (Kd = 1.5 nM) and low capacity (Bmax = 40 pmol/mg protein).
- Optimal binding required specific stereochemistry (5(S), 12(R) hydroxyls), a cis-double bond at C-6, and an intact eicosanoid backbone, correlating with agonistic activity.
Conclusions:
- High-affinity, stereoselective, and specific receptors for LTB4 exist in the human PMN plasma membrane.
- The binding characteristics of LTB4 analogs confirm the structural requirements for receptor activation.
- These findings provide a molecular basis for LTB4-mediated neutrophil functions in inflammation.