SUMMARY Interleukin-16 (IL-16) acts as a chemoattractant for CD4+ cells, as a modulator of T-cell activation, and plays a key role in asthma. This report describes the cytokine-inducing effects of IL-16 on total peripheral blood mononuclear cells (PBMC) and PBMC subpopulations. While CD4 + T lymphocytes did not secrete cytokines in response to rhIL-16, CD14+ CD4 + monocytes and maturing macrophages secrete IL-1b, IL-6, IL-15 and tumour necrosis factor-a (TNF-a) upon rhIL-16 stimulation. The mRNA species for these four cytokines were detected as early as 4 hr poststimulation, with protein being secreted by 24 hr. Secretion of IL-1b and IL-6 by total PBMC was dose dependent, with maximal secretion being observed using 50 ng/ml rhIL-16. However, for IL-15 or TNF-a maximal secretion by total PBMC occurred with all concentrations between 5 ng/ml to 500 ng/ml rhIL-16. Puri®ed monocytes/macrophages secreted maximal concentrations of all four cytokines in the presence of 500 ng/ml rhIL-16, except for monocytes where maximal secretion of IL-15 was, interestingly, observed with only 50 ng/ml rhIL-16. The use of higher concentrations of rhIL-16 (1000 ng/ml) inhibited secretion of all four cytokines. While these IL-16-induced cytokines are likely to be involved in the immune system's response to antigen, the data suggest that IL-16 may play a key role in initiating and/or sustaining an in¯ammatory response.

The N-terminal extracellular parts of human G-protein coupled receptor class B, for example, receptors for secretin, glucagon, or parathyroid hormone, are involved in ligand binding. To obtain structural and functional information on the N-terminal receptor fragment of human parathyroid hormone receptor 1 (PTHR1), the truncated receptor was expressed in the cytosol of Escherichia coli in the form of inclusion bodies. Oxidative refolding of inclusion body material resulted in stable, soluble, monomeric protein. Ligand binding was proved by surface plasmon resonance spectroscopy and isothermal titration calorimetry. Refolded receptor fragment was able to bind parathyroid hormone with an apparent dissociation constant of 3-5 microM. Far-UV circular dichroism spectra showed that the refolded polypeptide contained approximately 25% alpha-helical and 23% beta-sheet secondary structures. Analysis of the disulfide bond pattern of the refolded receptor fragment revealed disulfide bonds between Cys170 and Cys131, Cys148 and Cys108, and Cys117 and Cys48. These results demonstrate that the extracellular N-terminal domain of the parathyroid hormone receptor (PTHR1) possesses a well-defined, stable conformation, which shows a significant ligand binding activity.

Homologous desensitization of G proteincoupled receptors is thought to occur in several steps: binding of G protein-coupled receptor kinases (GRKs) to receptors, receptor phosphorylation, kinase dissociation, and finally binding of ␤-arrestins to phosphorylated receptors. It generally is assumed that only the last step inhibits receptor signaling. Investigating the parathyroid hormone (PTH) receptor 3 inositol phosphate pathway, we report here that GRKs can inhibit receptor signaling already under nonphosphorylating conditions. GRKs phosphorylated the PTH receptor in membranes and in intact cells; the order of efficacy was GRK2>GRK3>GRK5. Transient transfection of GRKs with the PTH receptor into COS-1 cells inhibited PTHstimulated inositol phosphate generation. Such an inhibition also was seen with the kinase-negative mutant GRK2-K220R and also for a C-terminal truncation mutant of the PTH receptor that could not be phosphorylated. Several lines of evidence indicated that this phosphorylation-independent inhibition was exerted by an interaction between GRKs and receptors: (a) this inhibition was not mimicked by proteins binding to G proteins, phosducin, and GRK2 C terminus, (b) GRKs caused an agonist-dependent inhibition ‫؍(‬ desensitization) of receptor-stimulated G protein GTPase-activity (this effect also was seen with the kinase-inactive GRK2-mutant and the phosphorylation-deficient receptor mutant), and (c) GRKs bound directly to the PTH receptor. These data suggest that signaling by the PTH receptor already is inhibited by the first step of homologous desensitization, the binding of GRKs to the receptors.

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