Assembly of G Protein-Coupled Receptors from Fragments:  Identification of Functional Receptors with Discontinuities in Each of the Loops Connecting Transmembrane Segments

Abstract

The alpha-factor receptor of the yeast Saccharomyces cerevisiae is a member of the superfamily of G protein-coupled receptors that mediate signal transduction in response to sensory and chemical stimuli. All members of this superfamily contain seven predicted transmembrane segments. We have created a series of genes encoding alpha-factor receptors with amino- or carboxyl-terminal truncations at each of the loop regions connecting transmembrane segments. Split receptors containing a discontinuity in the peptide backbone were synthesized by coexpressing pairs of truncated receptor fragments in yeast. Complementary pairs of fragments split at sites within each of the cytoplasmic and extracellular loops were capable of assembling and transducing a signal in response to alpha-factor binding. One pair of noncomplementary fragments containing a deletion in the second intracellular loop of the receptor also yielded a functional receptor. Coexpression of certain combinations of overlapping fragments containing supernumerary transmembrane segments also led to formation of functional receptors, apparently because of proteolytic trimming of overlapping regions. Coexpression of truncated receptor fragments with full-length receptors had no effect on signaling by the full-length receptors. These results demonstrate the following: (1) Correct folding of the alpha-factor receptor does not require a covalent connection between any pair of transmembrane segments that are adjacent in the sequence. (2) Most of the second intracellular loop of the receptor is not required for function. (3) The structure of the receptor cannot, in most cases, tolerate the presence of extra transmembrane segments. (4) None of the truncated fragments of the alpha-factor receptor can efficiently oligomerize with normal receptors in such a way as to inhibit receptor function.

A Microdomain Formed by the Extracellular Ends of the Transmembrane Domains Promotes Activation of the G Protein-Coupled α-Factor Receptor

“…In contrast, very few mutations that cause defects in Ste2p signaling have been identified in the outer-loop regions as a result of scanning-or random-mutagenesis strategies (1,9,29,30,38,48,55). The notion that the outer loops are not as critical for receptor activation is supported by the observation that functional ␣-factor receptors can be reconstituted from coexpressed halves of Ste2p that are split in the loop regions (32). These results indicate that residues in the membrane interface region play key functional roles in ligand binding and receptor activation.…”

The Chain Length Dependence of Helix Formation of the Second Transmembrane Domain of a G Protein-coupled Receptor ofSaccharomyces cerevisiae

“…judged by both CD and IR analyses. Three groups have reported hydropathy analyses on Ste2p (18,56,57). These analyses differ very slightly in the residues placed at the membrane interface and the number of residues actually in the bilayer.…”

“…These analyses differ very slightly in the residues placed at the membrane interface and the number of residues actually in the bilayer. Two of the analyses predicted 24 transmembrane residues in the second TMD (18,56), whereas the third predicted that only 21 residues of this domain would be in the bilayer. Given these predictions and assuming that the SNYSS sequence could enter the hydrophobic region of the bilayer, only M2-22, M2-26, M2-30, and M2-35 have sufficient residues to fully span the lipid.…”

“…The difference in the tilt angles of the four peptides that form helical structures in the vesicles and multilayers (M2-22, M2-26, M2-30B, and M2-35) may also be the result of hydrophobic mismatch. If one assumes that the hydropathy predictions are correct (18,56,57), then only about 17, 21, 25, and 25 residues, respectively, of these peptides should be in the hydrocarbon domain of the membrane. The SNYSS sequence should be outside the membrane core.…”

“…The finding again points to the relevance of results from studies in TFE water mixtures to the lipid milieu. In the case of M2-30, the peptide is predicted to have 4 -7 residues that are in the extracellular loop of Ste2p and 23-26 residues in the bilayer (18,56,57). It is likely that this span is too long for the bilayer, thereby causing a hydrophobic mismatch.…”