It is becoming increasingly clear that the autonomic nervous system and the immune system demonstrate cross-talk during inflammation by means of sympathetic and parasympathetic pathways. We investigated whether phagocytes are capable of de novo production of catecholamines, suggesting an autocrine/paracrine self-regulatory mechanism by catecholamines during inflammation, as has been described for lymphocytes. Here we show that exposure of phagocytes to lipopolysaccharide led to a release of catecholamines and an induction of catecholamine-generating and degrading enzymes, indicating the presence of the complete intracellular machinery for the generation, release and inactivation of catecholamines. To assess the importance of these findings in vivo, we chose two models of acute lung injury. Blockade of alpha2-adrenoreceptors or catecholamine-generating enzymes greatly suppressed lung inflammation, whereas the opposite was the case either for an alpha2-adrenoreceptor agonist or for inhibition of catecholamine-degrading enzymes. We were able to exclude T cells or sympathetic nerve endings as sources of the injury-modulating catecholamines. Our studies identify phagocytes as a new source of catecholamines, which enhance the inflammatory response.

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Despite extensive study of heptahelical G protein-coupled receptors (GPCRs), the precise mechanism of G protein activation is unknown. The role of one highly conserved stretch of residues, the amino acids glutamic acid/aspartic acid-argininetyrosine (i.e., the E/DRY motif), has received considerable attention with respect to regulating GPCR conformational states. In the consensus view, glutamic acid/aspartic acid maintains the receptor in its ground state, because mutations frequently induce constitutive activity (CA). This hypothesis has been confirmed by the rhodopsin ground-state crystal structure and by computational modeling approaches. However, some class A GPCRs are resistant to CA, suggesting alternative roles for the glutamic acid/aspartic acid residue and the E/DRY motif. Here, we propose two different subgroups of receptors within class A GPCRs that make different use of the E/DRY motif, independent of the G protein type (G s , G i , or G q ) to which the receptor couples. In phenotype 1 receptors, nonconservative mutations of the glutamic acid/aspartic acid-arginine residues, besides inducing CA, increase affinity for agonist binding, retain G protein coupling, and retain an agonist-induced response. In contrast, in second phenotype receptors, the E/DRY motif is more directly involved in governing receptor conformation and G protein coupling/recognition. Hence, mutations of the glutamic acid/aspartic acid residues do not induce CA. Conversely, nonconservative mutations of the arginine of the E/DRY motif always impair agonist-induced receptor responses and, generally, reduce agonist binding affinity. Thus, it is essential to look beyond the rhodopsin ground-state model of conformational activation to clarify the role of this highly conserved triplet in GPCR activation and function.

G-protein-coupled receptors (GPCRs) are major targets for drug discovery. The regulator of G-protein signalling (RGS)-protein family has important roles in GPCR signal transduction. RGS proteins contain a conserved RGS-box, which is often accompanied by other signalling regulatory elements. RGS proteins accelerate the deactivation of G proteins to reduce GPCR signalling; however, some also have an effector function and transmit signals. Combining GPCR agonists with RGS inhibitors should potentiate responses, and could markedly increase the agonist's regional specificity. The diversity of RGS proteins with highly localized and dynamically regulated distributions in brain makes them attractive targets for pharmacotherapy of central nervous system disorders.

The guanine nucleotide exchange factor p63RhoGEF is an effector of the heterotrimeric guanine nucleotide-binding protein (G protein) Galphaq and thereby links Galphaq-coupled receptors (GPCRs) to the activation of the small-molecular-weight G protein RhoA. We determined the crystal structure of the Galphaq-p63RhoGEF-RhoA complex, detailing the interactions of Galphaq with the Dbl and pleckstrin homology (DH and PH) domains of p63RhoGEF. These interactions involve the effector-binding site and the C-terminal region of Galphaq and appear to relieve autoinhibition of the catalytic DH domain by the PH domain. Trio, Duet, and p63RhoGEF are shown to constitute a family of Galphaq effectors that appear to activate RhoA both in vitro and in intact cells. We propose that this structure represents the crux of an ancient signal transduction pathway that is expected to be important in an array of physiological processes.

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