Assembly of G protein-coupled receptors from fragments: identification of functional receptors with discontinuities in each of the loops connecting transmembrane segments.
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.
Martin
N P
NP
Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, New York 14642, USA.
Leavitt
L M
LM
Sommers
C M
CM
Dumont
M E
ME
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism* / Q000502:physiology; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D017434:Protein Structure, Tertiary; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D012441:Saccharomyces cerevisiae; D017384:Sequence Deletion; D014157:Transcription Factors
D010446:Peptide Fragments; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D014157:Transcription Factors; D019204:GTP-Binding Proteins