Received September 9, 2002; Revised October 17 and November 15, 2002; Accepted November 15, 2002. Author to whom all correspondence and reprint requests should be addressed: Grace L. Rosenquist, Section of Neurobiology, Physiology and Behavior, University of California, 1 Shields Avenue, Davis, CA 95616. E-mail: rosenqui @psc.edu Prediction of Tyrosine Sulfation in Seven-Transmembrane Peptide Receptors Kristine M. Yu, 1 Justin Liu, 1 Ryan Moy, 1 Henry C. Lin, 1 Hugh B. Nicholas, Jr., 2 and Grace L. Rosenquist 1 1 Section of Neurobiology, Physiology and Behavior, University of California, Davis, CA; and 2 Pittsburgh Supercomputing Center, Mellon Institute, 4400 Fifth Avenue, Pittsburgh, PA Endocrine, vol. 19, no. 3, 333–337, December 2002 0969–711X/02/19:333–337/$20.00 © 2002 by Humana Press Inc. All rights of any nature whatsoever reserved. 333 Posttranslational modification by tyrosine sulfation reg- ulates many important protein–protein interactions and modulates the binding affinity and specificity of seven- transmembrane peptide receptors. We developed a log-odds position-specific-scoring-matrix (PSSM) to accurately predict tyrosine sulfation using 62 tyrosine sites known to be sulfated and 421 tyrosine sites known not to be sulfated. We predict that 49 tyrosines of 32 seven-transmembrane peptide receptors are sulfated. Although we did not incorporate characteristics of con- firmed sulfation sites such as clustering and conser- vation across species into our PSSM, our predicted sites nevertheless exhibited these characteristics. The observed conservation suggests that there are strong evolutionary pressures to preserve selected biological activity of seven-transmembrane receptors. The pre- dicted tyrosine sulfation sites predominantly occur in the extracellular tail and extracellular loop 2, regions consistent with their association with binding pockets of the receptor. Key Words: Tyrosine sulfation; posttranslational process- ing; seven-transmembrane receptors; G-coupled pro- tein receptors; peptides. Introduction Tyrosine sulfation, catalyzed by tyrosylprotein sulfo- transferases (TPSTs), is the most common posttranslational modification of tyrosine residues transported through the Golgi system in eukaryotes (1,2). The importance of tyro- sine sulfation became widely recognized in 1999 after the demonstration of sulfation in the seven-transmembrane (7TM) receptor CCR5 (3,4). Recent studies have strongly sup- ported that tyrosine sulfation is also required for optimal protein–protein interactions and specific functions of many other 7TM receptors, such as CCR2 (5), CX3CR1 (6), C5aR (7), and CXCR4 (8). While information on tyrosine sulfation of chemokine and chemotactic factor receptors is well established, knowl- edge about tyrosine sulfation in 7TM peptide receptors is more preliminary. Currently, only the thyrotropin receptor (TSHr) has been directly shown to be tyrosine sulfated, although experimental evidence suggests that the luteiniz- ing hormone receptor (LHr) and the follicle-stimulating receptor (FSHr) are likely to be tyrosine sulfated as well (9). However, tyrosine sulfation of other 7TM peptide recep- tors has not been reported. While the impact of tyrosine sul- fation on 7TM peptide receptors remains to be described, this posttranslational modification may be involved with many of the functions associated with regulatory peptide receptors, such as anti-inflammation, glucose metabolism, food intake, and the response to stress. Thus, the identifi- cation of tyrosine sulfation in 7TM peptide receptors may expedite the study of 7TM peptide receptors and elucidate the nature of their interactions with other ligands and proteins. In our study, a position-specific-scoring-matrix (PSSM) was used to predict the sulfation of 49 tyrosines of 32 7TM peptide receptors. These predictions are consistent with what is known about tyrosine sulfation. Most notably, the loca- tions of the predicted sulfated tyrosines in the receptors cor- respond to those of experimentally confirmed sites. Results Forty-nine tyrosines of 32 receptors were predicted to be sulfated out of a total of 756 tyrosine sites (Table 1). Out of these 49 predicted tyrosines, 36 (73.5%) are located in the N-terminal extracellular tail of the receptors or in the second extracellular loop between transmembrane helices 4 and 5 (Fig. 1). In addition, a few sites in the first and third extracellular loops were also predicted to be sulfated. Unex- pectedly, five predicted tyrosine sulfation sites are located in the cytoplasmic tail. When multiple tyrosines of a receptor were predicted to be sulfated, they were often located in proximity to each other. For instance, the corticotropin-releasing-factor (CRF) recep- tor 1, FMLP-related, FSH, glucagon-like peptide (GLP), and LSH receptors possess clusters of predicted sulfated tyrosines located within five residues of each other (Table 1, highlighted). This clustering has previously been noted in other experimentally determined tyrosine sulfation sites.