A Dual Receptor Crosstalk Model of G-Protein-Coupled Signal Transduction

Macrophage cells that are stimulated by two different ligands that bind to G-protein-coupled receptors (GPCRs) usually respond as if the stimulus effects are additive, but for a minority of ligand combinations the response is synergistic. The G-protein-coupled receptor system integrates signaling cues from the environment to actuate cell morphology, gene expression, ion homeostasis, and other physiological states. We analyze the effects of the two signaling molecules complement factors 5a (C5a) and uridine diphosphate (UDP) on the intracellular second messenger calcium to elucidate the principles that govern the processing of multiple signals by GPCRs. We have developed a formal hypothesis, in the form of a kinetic model, for the mechanism of action of this GPCR signal transduction system using data obtained from RAW264.7 macrophage cells. Bayesian statistical methods are employed to represent uncertainty in both data and model parameters and formally tie the model to experimental data. When the model is also used as a tool in the design of experiments, it predicts a synergistic region in the calcium peak height dose response that results when cells are simultaneously stimulated by C5a and UDP. An analysis of the model reveals a potential mechanism for crosstalk between the Gαi-coupled C5a receptor and the Gαq-coupled UDP receptor signaling systems that results in synergistic calcium release

On the structure of the amino-terminal domain ED 1 of the B2 receptor

Receptors for kinins are classified into B 1 (Menke et al., 1994) and B2 (McEachern et al., 1991) sub- types. Both receptors belong to the seven transmembrane domain (TMD) receptor family which activate trimeric G-proteins. The cDNAs coding for the rat (McEachern et al., 1991), the human (Hess et al., 1992; Eggerickx et al., 1992) and the mouse (Yokoyama et al., 1994) B2 receptors contain several in-frame methionine codons which could be used as an initiation site for translation; however, none of them conforms to the 'canonical' consensus sequence often preceding the initiation codon (Kozak, 1989). On the basis of sequence similarity studies the third in-frame AUG of the human B2 receptor mRNA, starting at nucleotide 223 of clone CCD-16- 2, was assumed to be the preferred start codon (Hess et al., 1992). The underlying gene of the human B2 receptor consists of three exons interrupted by two introns (Ma et al., 1994): exon-1 is non-coding, the first two in-frame AUG codons are located on exon-2, and the third in-frame AUG is located on exon-3 together with the seven transmembrane spanning sequence segments. There is no evidence for alternative splicing of the human B2 receptor pre-mRNA (Kammerer et al., 1995). To clarify which in-frame AUG serves as the translation initiation codon, we have isolated the B2 receptor protein and determined its amino-terminal sequence (Abd Alia et al., in preparation). The sequence data indicate that the B2 receptor mRNA from human foreskin fibroblasts is translated from the first in-frame initiator AUG codon. We report here that the extra segment of 27 amino acid residues which precedes the predicted amino-terminus of the receptor protein does not significantly alter the binding characteristics of the receptor

Crosstalk between GABAB and mGlu1a receptors reveals new insight into GPCR signal integration

G protein-coupled receptors (GPCRs) have critical functions in intercellular communication. Although a wide range of different receptors have been identified in the same cells, the mechanism by which signals are integrated remains elusive. The ability of GPCRs to form dimers or larger hetero-oligomers is thought to generate such signal integration. We examined the molecular mechanisms responsible for the GABAB receptor-mediated potentiation of the mGlu receptor signalling reported in Purkinje neurons. We showed that this effect does not require a physical interaction between both receptors. Instead, it is the result of a more general mechanism in which the βγ subunits produced by the Gi-coupled GABAB receptor enhance the mGlu-mediated Gq response. Most importantly, this mechanism could be generally applied to other pairs of Gi- and Gq-coupled receptors and the signal integration varied depending on the time delay between activation of each receptor. Such a mechanism helps explain specific properties of cells expressing two different Gi- and Gq-coupled receptors activated by a single transmitter, or properties of GPCRs naturally coupled to both types of the G protein