We describe a procedure for isolating agonists for mammalian G protein-coupled receptors of unknown function. Human formyl peptide receptor like-1 (FPRL-1) receptor, originally identified as an orphan G protein-coupled receptor related to the formyl peptide receptor (FPR1), was expressed in Saccharomyces cells designed to couple receptor activation to histidine prototrophy. Selection for histidine prototrophs among transformants obtained with a plasmid-based library encoding random peptides identified six different agonists, each of whose production yielded autocrine stimulation of the receptor expressed in yeast. A synthetic version of each peptide promoted activation of FPRL-1 expressed in human embryonic kidney (HEK293) cells, and five of the peptides exhibited significant selectivity for activation of FPRL-1 relative to FPR1. One selective peptide was tested and found to mobilize calcium in isolated human neutrophils. This demonstrates that stimulation of FPRL-1 results in neutrophil activation and suggests that the receptor functions as a component of the inflammatory response. This autocrine selection protocol may be a generally applicable method for providing pharmacological tools to evaluate the physiological roles of the growing number of mammalian orphan G protein-coupled receptors.

Multiple different subunits of fibronectin are known to occur and their origin has been unclear. Recent results showing that a single fibronectin gene can give rise to se%'eral different mRNAs by alternative splicing suggested an explanation for some of this diversity of fibronectin subunits. Because the alternative splicing events occur within the coding region, the mRNAs differ in coding potential. We have prepared recombinant phage containing a rat fibronectin cDNA segment that is present in some fibronectin mRNAs and not in others. This segment was inserted in the 13-galatosidase gene of )Xgt11, and fusion protein produced by lysogens of the recombinant phage was purified and used as immunogen. The resulting antisera recognized some subunits of rat and hamster fibronectins but not others, indicating that inclusion or removal of this segment gives rise to mRNAs that encode different fibronectin subunits. In particular, presence or absence of a 95 amino acid segment appears to account for differences in size among the subunits of plasma fibronectin, whose origin is therefore explained by alternative patterns of RNA splicing.Fibronectins (FNs) comprise a group of large extracellular glycoproteins composed of structurally similar but nonidentical subunits varying in size between 210 and 250 kDa (1-3). One-and two-dimensional electrophoresis have shown that each form of FN consists of a characteristic set of distinguishable subunits (4-7). For example, one form of FN, pFN, which is found in blood plasma, consists of four types of subunit which fall into two size classes differing in apparent molecular mass by about 10 kDa. The FN synthesized by fibroblasts (cellular or cFN) contains subunits not detected in pFN (6,7). This diversity of subunits requires an explanation. Investigations of the known posttranslational modifications (7) suggest that these modifications cannot account for all the heterogeneity, raising the possibility that the subunits differ in primary sequence. Such differences could, in principle, arise from the existence of multiple genes, from alternative processing of the primary transcript of a single gene, from posttranslational cleavages, or from some combination of these mechanisms.We have recently isolated cDNA (8) and genomic clones (9) encoding rat FNs and have shown that several different mRNAs arise by alternative splicing of the transcript of a single FN gene. Three mRNA species were found in rat liver, the source of pFN. These mRNAs differ at a point 827 bases before the termination codon by the inclusion or omission of blocks of coding sequence, 285 or 360 (285 + 75) bases long (8). Therefore, these mRNAs encode polypeptides that differ by 95 or 120 amino acids. It seemed probable that these differences could account for some of the known FN subunits. We report here the preparation of an antiserum against the 95 amino acid segment and its use to demonstrate that this segment is present in the larger subunits of pFN but absent from the smaller ones.MATERIALS AND METHODS Cel...

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