Signal Transduction by the Chemokine Receptor CXCR5: Structural Requirements for G Protein Activation Analyzed by Chimeric CXCR1/CXCR5 Molecules vating heterotrimeric G proteins (reviewed in Wess, 1997). The latter initiate various intracellular signaling pathways by modulating the activity of effectors, such as enzymes, ion channels and transporters, thereby influ- encing the concentration of second messengers. Apart from G proteins, only a few other proteins are known to directly interact with GPCRs at the cytoplasmic surface of the molecules. These are usually regulatory factors modulating signal transduction, e. g. by controlling re- ceptor desensitization and internalization (reviewed in Böhm et al., 1997). Based on the position of conserved cysteine residues in their primary amino acid sequence, chemokines are grouped into four subfamilies: C, CC, CXC, and CX 3 C chemokines. Accordingly, chemokine re- ceptors are assigned to corresponding subsets. This classification takes into account that, although binding of a given chemokine is not restricted to one distinct GPCR (and vice versa), such interaction usually occurs between molecules belonging to the corresponding groups. CXC-chemokine receptor 1 (CXCR1), one of the two known human receptors for interleukin-8 (IL-8/CXCL8), a major mediator for inflammatory reactions, is one of the best-characterized chemokine receptors to date (re- viewed in Baggiolini et al., 1994, 1997). Activation of CXCR1 by CXCL8 gives rise to an increase in intracellular calcium (Jones et al., 1996), stimulation of phospholipase C (PLC), and phospholipase D (PLD) (Wu et al., 1993; Jones et al., 1996), as well as the activation of the extracel- lular signal-regulated kinase/mitogen-activated protein ki- nase (ERK/MAPK) pathway (Jones et al., 1995). Moreover, stimulation with CXCL8 induces chemotaxis of either neu- trophils or cells ectopically expressing CXCR1 (Walz et al., 1987; Yoshimura et al., 1987; Loetscher et al., 1994), the adhesion of monocytes to vascular endothelium express- ing E-selectin and of CXCR1-transfected mouse L1/2 cells to VCAM-1 coated surfaces under flow conditions (Campbell et al., 1996; Gerszten et al., 1999). In addition, CXCL8 has been shown to trigger the transendothelial mi- gration of neutrophils (Huber et al., 1991). CXCR1 has also been extensively characterized on the molecular level. Amino acids essential for ligand binding, the interaction with heterotrimeric G proteins, receptor phosphorylation and desensitization as well as phosphoinositide hydroly- sis have been mapped by receptor mutagenesis (Hebert et al., 1993; Leong et al., 1994; Richardson et al., 1995; Damaj et al., 1996; Xie et al., 1997). In contrast to the detailed observations regarding the molecular biology of CXCR1, only few information is avail- able on signaling via the CXC-chemokine receptor 5 Biol. Chem., Vol. 382, pp. 1387 – 1397, September 2001 · Copyright © by Walter de Gruyter · Berlin · New York Gerd Müller and Martin Lipp* Department of Molecular Tumor Genetics and Immunogenetics, Max-Delbrück-Center for Molecular Medicine, D-13092 Berlin, Germany * Corresponding author The human chemokine receptors CXCR5 and CXCR1 activate signaling pathways via pertussis toxin-sensi- tive as well as insensitive G proteins. CXCR5 induces Ca 2+ signaling and chemotaxis independently of in- hibitory G proteins, whereas the same signaling path- ways are entirely dependent on inhibitory G proteins for CXCR1. In contrast, activation of the MAP kinase cascade via ERK1/2 is a pertussis toxin-sensitive signaling event for both receptors. Using chimeric CXCR1/CXCR5 receptors we investigated structural requirements for the activation of signal transduction pathways by CXCR5. Individual or multiple intracellular domains of CXCR1 were exchanged for the correspon- ding sequences of CXCR5, leading to receptors resem- bling CXCR5 at the cytoplasmic surface to a varying ex- tent. Replacing the second intracellular domain of CXCR1 had a major influence on signaling mediated by inhibitory G proteins, whereas the exchange of the third or carboxy-terminal intracellular domain had only mi- nor effects on signal transduction. Activation of the MAP kinase cascade via ERK1/2 and chemotaxis are largely reduced in chimeras comprising the second in- tracellular domain of CXCR5, although coupling to in- hibitory G proteins is retained in all chimeric receptors. In summary, these data characterize the contribution of the intracellular domains of CXCR5 to receptor signal- ing, thereby disclosing unique structural requirements that modulate G protein coupling by the receptor. Key words: Chemokine / CXCL8 / CXCL13 / CXCR1 / CXCR5. Introduction Chemokine receptors belong to the superfamily of G pro- tein-coupled receptors (GPCRs), sharing the structural hallmark of seven transmembrane spanning domains (TM) alternately connected by loops located in the cyto- plasmic and extracellular region (Figure 1). Binding of a distinct agonist to its cognate GPCR is believed to evoke structural rearrangements of the receptor, thereby acti- Brought to you by | Purdue University Libraries Authenticated Download Date | 6/14/15 9:34 PM