Dominant-Negative Mutations in the G-Protein-Coupled α-Factor Receptor Map to the Extracellular Ends of the Transmembrane Segments

Abstract

G-protein-coupled receptors (GPCRs) transduce the signals for a wide range of hormonal and sensory stimuli by activating a heterotrimeric guanine nucleotide-binding protein (G protein). The analysis of loss-offunction and constitutively active receptor mutants has helped to reveal the functional properties of GPCRs and their role in human diseases. Here we describe the identification of a new class of mutants, dominantnegative mutants, for the yeast G-protein-coupled ␣-factor receptor (Ste2p). Sixteen dominant-negative receptor mutants were isolated based on their ability to inhibit the response to mating pheromone in cells that also express wild-type receptors. Detailed analysis of two of the strongest mutant receptors showed that, unlike other GPCR interfering mutants, they were properly localized at the plasma membrane and did not alter the stability or localization of wild-type receptors. Furthermore, their dominant-negative effect was inversely proportional to the relative amount of wild-type receptors and was reversed by overexpressing the G-protein subunits, suggesting that these mutants compete with the wild-type receptors for the G protein. Interestingly, the dominant-negative mutations are all located at the extracellular ends of the transmembrane segments, defining a novel region of the receptor that is important for receptor signaling. Altogether, our results identify residues of the ␣-factor receptor specifically involved in ligand binding and receptor activation and define a new mechanism by which GPCRs can be inactivated that has important implications for the evaluation of receptor mutations in other G-protein-coupled receptors.G-protein-coupled receptors (GPCRs) comprise a large family of receptors that are found in a wide range of eukaryotic organisms from yeasts to humans (4, 10). These receptors respond to diverse stimuli including hormones, neurotransmitters, and other chemical messengers (48). GPCRs transduce their signal by stimulating the ␣ subunit of a heterotrimeric guanine nucleotide binding protein (G protein) to bind GTP (4, 16). This releases the ␣ subunit from the ␤␥ subunits, and then either the ␣ subunit or the ␤␥ subunits go on to promote signaling depending on the specific pathway (28).GPCRs are structurally similar in that they contain seven transmembrane domains (TMDs) connected by intracellular and extracellular loops. Although many techniques have been applied to study receptor function, much of our knowledge on the mechanisms of GPCR activation comes from the characterization of mutant receptors. Loss-of-function and supersensitive mutants have helped to identify receptor regions needed for ligand binding, G-protein activation, and down-regulation of signaling (4, 49). Furthermore, the study of constitutively active receptor mutations has played a key role in the development of current models for receptor activation (26). Naturally occurring GPCR mutations have also been implicated in a number of human diseases (8,25,42). Interestingly, the analysis of different mutant rec...

Secretin Receptor Oligomers Form Intracellularly during Maturation through Receptor Core Domains

“…However, defects in plasma membrane sorting are not an inherent characteristic of oligomerization-related phenotypes, because both misspliced and wild type secretin receptors sorted to and colocalized on the surfaces of COS and Panc-1 cells (26,28). Consistent with our previous hypotheses, oligomerization thus provides a structural mechanism for how misspliced secretin receptor inhibits wild type receptor function, perhaps by changing the ability of wild type receptors to compete for G protein coupling at the cell surface (32). In conclusion, these initial assessments, and this study's findings in general, are important contributions to our understanding of the mechanism of secretin receptor association and its implications for receptor function and disease progression.…”

Constitutively Active G Protein-coupled Receptor Mutants BlockDictyosteliumDevelopment

“…Our DN mutants appear not to act by either of these mechanisms because they are expressed efficiently on the cell surface and DN4⌬P did not alter either the surface localization or cAMP-induced phosphorylation of coexpressed wild-type cAR1. In addition, G protein-sequestering mutants of Ste2 and mammalian ␣ 1B -adrenergic receptor have been described (Dosil et al, 1998;Chen et al, 2000). Although we have not formally ruled out this mechanism for our DN mutants, it seems unlikely as it does not readily account for their constitutive phosphorylation.…”

Semidominant Mutations in Reduced Epidermal Fluorescence 4 Reduce Phenylpropanoid Content in Arabidopsis

“…The wildtype function of REF4 could be to attenuate this signaling in response to external cues, with the dominant mutations preventing modulation of this effect, leading to constitutive downregulation of phenylpropanoid metabolism. Interestingly, dominant mutations in a G-protein-coupled receptor are primarily the result of amino acid substitutions in, or immediately adjacent to, TMRs (Dosil et al 1998), as is the case in mutant REF4 alleles. If G-protein-coupled receptors can serve as a model for REF4, missense mutations in REF4 could lock the encoded protein into a constitutively active state.…”