Selective Modulation of Wild Type Receptor Functions by Mutants of G-Protein-coupled Receptors

Abstract

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...

Discovery of Naturally Occurring Splice Variants of the Rat Histamine H3Receptor That Act as Dominant-Negative Isoforms

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Oligomeric potential of the M2 muscarinic cholinergic receptor

“…This is suggested by evidence that the trafficking of wild-type receptors to the cell surface can be inhibited by mutants (e.g. Le Gouill et al 1999;Zhu and Wess 1998); also, the transfer of resonance energy between tagged receptors can be detected in membrane fractions enriched in endoplasmic reticulum (Terrillon et al 2003). It follows that the oligomeric status of the receptor is established early on and therefore is expected to be the same regardless of whether the receptor is localized intracellularly or at the plasma membrane.…”

Oligomerization of Wild Type and Nonfunctional Mutant Angiotensin II Type I Receptors Inhibits Gαq Protein Signaling but Not ERK Activation

“…1) The mutant receptor does not affect wild type signaling; 2) the mutant receptor inhibits wild type signaling either by inhibiting surface expression or the activation of signaling (8 -10); or 3) the wild type receptor can functionally rescue the mutant receptor (11,12,37). In addition, co-expression of two nonfunctional receptors can create functional receptors (17,38).…”