A Chemoattractant-mediated G_i-coupled Pathway Activates Adenylyl Cyclase in Human Neutrophils

Abstract

Neutrophils and Dictyostelium use conserved signal transduction pathways to decipher chemoattractant gradients and migrate directionally. In both cell types, addition of chemoattractants stimulates the production of cAMP, which has been suggested to regulate chemotaxis. We set out to define the mechanism by which chemoattractants increase cAMP levels in human neutrophils. We show that chemoattractants elicit a rapid and transient activation of adenylyl cyclase (AC). This activation is sensitive to pertussis toxin treatment but independent of phosphoinositide-3 kinase activity and an intact cytoskeleton. Remarkably, and in sharp contrast to G␣ s -mediated activation, chemoattractant-induced AC activation is lost in cell lysates. Of the nine, differentially regulated transmembrane AC isoforms in the human genome, we find that isoforms III, IV, VII, and IX are expressed in human neutrophils. We conclude that the signal transduction cascade used by chemoattractants to activate AC is conserved in Dictyostelium and human neutrophils and is markedly different from the canonical G␣ s -meditated pathway. INTRODUCTIONMany types of cells have the ability to migrate directionally when exposed to gradients of chemoattractants. This chemotactic response is essential for a variety of physiological processes and is initiated when chemoattractants bind surface receptors and activate a wide range of signal transduction cascades, which ultimately lead to cellular polarization and migration (Parent, 2004). The acquisition of polarity is accompanied by a dramatic redistribution of cytoskeletal components, where F-actin and numerous actin-binding proteins are enriched at the front or leading edge and myosin II is assembled on the sides and at the back or trailing edge (Van Haastert and Devreotes, 2004;Bagorda et al., 2006).A striking chemotactic behavior is exhibited by neutrophils as they move across vascular barriers and navigate to sites of inflammation (Niggli, 2003b;Parent, 2004). The agents that induce directed migration of neutrophils include a large and diverse group of chemoattractants originating from different sources (Uhing and Snyderman, 1999). These agents include formylated peptides secreted by bacteria (such as N-formylmethionyl-leucyl-phenylalanine [fMLP]), products of the complement cascade (such as complement factor 5a [C5a]), and phospholipid metabolites (such as leukotriene B4 [LTB 4 ]) as well as a large family of chemokines that are derived from endothelial, epithelial, and stromal cells (Baggiolini, 1998). Remarkably, neutrophils can also relay the signal to surrounding cells by stimulating the production and release of more attractants, such as LTB 4 and interleukin (IL)-8, which act in a paracrine manner to spread the chemotactic response to surrounding cells (Bazzoni et al., 1991;Baggiolini et al., 1994;Kannan, 2002). Chemoattractants bind to serpentine transmembrane receptors that couple to heterotrimeric G proteins (Murphy, 1996). Receptor activation leads to the dissociation of the G protein into ␣-and...

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