Abstract
G protein-coupled receptors (GPCRs) can form dimeric or oligomeric complexes in vivo. However, the functions and mechanisms of oligomerization remain poorly understood for most GPCRs, including the alpha-factor receptor (STE2 gene product) of the yeast Saccharomyces cerevisiae. Here we provide evidence indicating that alpha-factor receptor oligomerization involves a GXXXG motif in the first transmembrane domain (TM1), similar to the transmembrane dimerization domain of glycophorin A. Results of fluorescence resonance energy transfer, fluorescence microscopy, endocytosis assays of receptor oligomerization in living cells, and agonist binding assays indicated that amino acid substitutions affecting the glycine residues of the GXXXG motif impaired alpha-factor receptor oligomerization and biogenesis in vivo but did not significantly impair agonist binding affinity. Mutant receptors exhibited signaling defects that were not due to impaired cell surface expression, indicating that oligomerization promotes alpha-factor receptor signal transduction. Structure-function studies suggested that the GXXXG motif in TM1 of the alpha-factor receptor promotes oligomerization by a mechanism similar to that used by the GXXXG dimerization motif of glycophorin A. In many mammalian GPCRs, motifs related to the GXXXG sequence are present in TM1 or other TM domains, suggesting that similar mechanisms are used by many GPCRs to form dimers or oligomeric arrays.
Highlights
G protein-coupled receptors (GPCR),1 which mediate the biological effects of many hormones, neurotransmitters, chemokines, and sensory stimuli, are the largest class of membranebound receptors
In many mammalian GPCRs, motifs related to the GXXXG sequence are present in TM1 or other transmembrane segments (TM) domains, suggesting that similar mechanisms are used by many GPCRs to form dimers or oligomeric arrays
A Glycophorin A-like Dimerization Motif in TM1 of the ␣-Factor Receptor—Because our previous studies indicated that TM1 is critical for ␣-factor receptor oligomerization [26], the purpose of the present study was to elucidate the mechanism by which TM1 promotes oligomerization by identifying point mutations that affect oligomerization in vivo
Summary
G protein-coupled receptors (GPCR), which mediate the biological effects of many hormones, neurotransmitters, chemokines, and sensory stimuli, are the largest class of membranebound receptors. TM1 may provide a dimer or oligomer contact site, as suggested by computational studies of -opioid receptors [22] and atomic force microscopy and surface-area modeling of rhodopsin [23]. A variation of this mechanism has been suggested by atomic force microscopy and modeling studies of rhodopsin, which suggest that TM4/5 forms the dimer interface and TM1 is used to assemble dimers into oligomeric arrays [23]. Taken together, these findings suggest that GPCRs of different classes may be segregated into separate arrays because different TM domain contacts are used to stabilize dimer and oligomer interfaces. ␣-Factor Receptor Oligomerization, Biogenesis, and Signaling oligomerization interface, which may involve the extracellular N terminus and TM2 [26]
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