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 tran sduction in response to sensory and chemical stimuli. All members of this s uperfamily contain seven predicted transmembrane segments. We have created a series of genes encoding alpha-factor receptors with amino- or carboxyl-t erminal truncations at each of the loop regions connecting transmembrane se gments. Split receptors containing a discontinuity in the peptide backbone were synthesized by coexpressing pairs of truncated receptor fragments in y east. Complementary pairs of fragments split at sites within each of the cy toplasmic and extracellular loops were capable of assembling and transducin g a signal in response to alpha-factor binding. One pair of noncomplementar y fragments containing a deletion in the second intracellular loop of the r eceptor also yielded a functional receptor. Coexpression of certain combina tions of overlapping fragments containing supernumerary transmembrane segme nts also led to formation of functional receptors, apparently because of pr oteolytic trimming of overlapping regions. Coexpression of truncated recept or fragments with Full-length receptors had no effect on signaling by the f ull-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 sequen ce. (2) Most of the second intracellular loop of the receptor is not requir ed for function. (3) The structure of the receptor cannot, in most cases, t olerate the presence of extra transmembrane segments. ( 1) None of the trun cated fragments of the alpha-factor receptor can efficiently oligomerize wi th normal receptors in such a way as to inhibit receptor function.