Complete mutagenesis of the extracellular domain of interleukin-8 (IL-8) type A receptor identifies charged residues mediating IL-8 binding and signal transduction.

We systematically converted each of the amino acids in the extracellular domain of the interleukin-8 (IL-8) type A receptor to alanine for the purpose of identifying amino acids contributing to IL-8 binding and IL-8-mediated signal transduction. We identified 20 mutations which cause a decrease in receptor affinity from a Kd of 2 nM to a Kd > or = 25 nM. We then analyzed these receptor mutants for their ability to mobilize intracellular calcium upon stimulation with 10 nM IL-8. The majority of the mutants were able to produce calcium fluxes at levels approximating that of wild-type IL-8 receptor A, with the exception of six mutants (R199A, R203A, C30A, C110A, C187A, and C277A) which showed no significant response. In addition, we performed calcium mobilization experiments to further characterize a series of previously constructed mutants which had only been characterized by their binding affinities in our previous report and found that mutant D265A showed no response upon stimulation with 10 nM IL-8. Our study shows that, besides the extracellular domain cysteines which may be critical for the overall folding of the receptor, three residues, Arg-199, Arg-203, and Asp-265, are important for IL-8 binding and IL-8-mediated signal transduction.

FIZZ1, a novel cysteine-rich secreted protein associated with pulmonary inflammation, defines a new gene family.

Bronchoalveolar lavage fluid from mice with experimentally induced allergic pulmonary inflammation contains a novel 9.4 kDa cysteine-rich secreted protein, FIZZ1 (found in inflammatory zone). Murine (m) FIZZ1 is the founding member of a new gene family including two other murine genes expressed, respectively, in intestinal crypt epithelium and white adipose tissue, and two related human genes. In control mice, FIZZ1 mRNA and protein expression occur at low levels in a subset of bronchial epithelial cells and in non-neuronal cells adjacent to neurovascular bundles in the peribronchial stroma, and in the wall of the large and small bowel. During allergic pulmonary inflammation, mFIZZ1 expression markedly increases in hypertrophic, hyperplastic bronchial epithelium and appears in type II alveolar pneumocytes. In vitro, recombinant mFIZZ1 inhibits the nerve growth factor (NGF)-mediated survival of rat embryonic day 14 dorsal root ganglion (DRG) neurons and NGF-induced CGRP gene expression in adult rat DRG neurons. In vivo, FIZZ1 may modulate the function of neurons innervating the bronchial tree, thereby altering the local tissue response to allergic pulmonary inflammation.