No abstract

COS-7 cells transfected with the leukotriene (LT) B4 receptor (BLTR) cDNA were unable to produce LTB4-induced inositol phosphates (IPs) in spite of the presence of endogenous Galphai, Galphaq and Galpha11 proteins. Co-transfection of BLTR with Galpha16, however, resulted in high levels of IP production, which were 17-, 10- and 6-fold higher than with co-transfected Galpha11, Galphaq and Galpha14, respectively. Co-transfection of BLTR with phospholipase C (PLC) beta2, on the other hand, resulted in efficient IP production and co-transfection of BLTR with both Galpha16 and PLCbeta2 resulted in a greater than additive response.

Members of the G-protein-coupled receptor (GPCR)family are involved in most aspects of higher eukaryote biology, and mutations in their coding sequence have been linked to several diseases. In the present study, we report that mutant GPCR can affect the functional properties of the co-expressed wild type (WT) receptor. Mutants of the human platelet-activating factor receptor that fail to show any detectable ligand binding (N285I and K298stop) or coupling to a G-protein (D63N, D289A, and Y293A) were co-expressed with the WT receptor in Chinese hamster ovary and COS-7 cells. In this context, N285I and K298stop mutant receptors inhibited 3 H-WEB2086 binding and surface expression. Co-transfection with D63N resulted in a constitutively active receptor phenotype. Platelet-activating factor-induced inositol phosphate production in cells transfected with a 1:1 ratio of WT:D63N was higher than with the WT cDNA alone but was abolished with a 1:3 ratio. We confirmed that these findings could be extended to other GPCRs by showing that co-expression of the WT C-C chemokine receptor 2b with a carboxyl-terminal deletion mutant (K311stop), resulted in a decreased affinity and responsiveness to MCP-1. A better understanding of this phenomenon could lead to important tools for the prevention or treatment of certain diseases.Certain ligands can assume distinct functions in different tissues. For example, platelet-activating factor (PAF) 1 is involved in embryogenesis as well as in modulation of a variety of functions of the immune and central nervous systems (1-3). With respect to PAF, for which the only known receptor is a member of the G-protein-coupled receptor (GPCR) family, the diversity of responses generated is likely a result of the receptor coupling to different signaling pathways in the target cells (3, 4). On the other hand, for other ligands such as adrenaline and dopamine, the cell can also determine the specificity of its response by the differential expression of receptor subtypes with distinct characteristics of binding, coupling, and desensitization (5). Subtypes of a given receptor can be derived from distinct genes or generated by alternative splicing. Divergence between receptor isoforms, as for the prostaglandin (EP) and the MCP-1 (CCR2) receptors, is most often limited to the carboxyl-terminal cytoplasmic tail, a region that is potentially involved in G-protein coupling, internalization, and down-regulation of the receptors (6, 7).Alternative splicing can also lead to the formation of nonfunctional receptors or receptors with certain functions that are greatly modified (8, 9). Individually, these receptors are not involved in signaling, but some of them can show dominantnegative properties when co-expressed with a functional subtype. It has recently been demonstrated that the expression of a truncated isoform of the human gonadotropin-releasing hormone receptor could affect the extent of agonist-specific cellular response by inhibiting the cell surface expression of the functional isoform (10). A similar e...

Mutational analysis of determinants located in the C-terminal (C) tail of the high affinity leukotriene (LT)BA variety of stimuli, including light, neurotransmitters, hormones, and inflammatory lipid mediators produce their effects via activation of G-protein-coupled receptors (GPCRs). 1 Taking into account the heterogeneity of ligands, it is interesting that all members of this superfamily of receptors share the same topology characterized by the presence of seven transmembrane ␣-helices. Conserved residues within subfamily A (related to rhodopsin) are localized throughout these helices, serving as reference points for sequence alignments. Biophysical and biochemical evidence indicate that upon photoactivation, relative movements of transmembrane (TM) 3 and TM6 occur in rhodopsin; similarly, rearrangement of the cytoplasmic proximal portion of TM7 relative to TM1 and of TM2 relative to the intracellular helix VIII were also observed (reviewed in Ref.