332 Ann. N.Y. Acad. Sci. 1041: 332–337 (2005). © 2005 New York Academy of Sciences. doi: 10.1196/annals.1282.053 Responses of GPCR135 to Human Gene 3 (H3) Relaxin in CHO-K1 Cells Determined by Microphysiometry EMMA T. VAN DER WESTHUIZEN, a,b PATRICK M. SEXTON, b ROSS A. D. BATHGATE, b AND ROGER J. SUMMERS a a Department of Pharmacology, Monash University, Clayton, Victoria 3168, Australia b Howard Florey Institute, University of Melbourne, Victoria 3010, Australia ABSTRACT: This study examined the functional response to human relaxin 2 (H2 relaxin), human relaxin 3 (H3 relaxin), porcine relaxin, and human INSL3 in the cytosensor microphysiometer, using CHO-K1 cells stably expressing hu- man GPCR135. CHO-K1 cells stably expressing GPCR135 were generated by the serial dilution method and receptor properties were assessed. Saturation studies of [ 125 I] H3 relaxin binding to GPCR135 in these cells gave a B max of 32.61 ± 6.5 fmol/mg protein and K d of 0.12 ± 0.08 nM. The functional response to H3 relaxin and other relaxin/insulin peptides of GPCR135 expressed in CHO-K1 cells was measured in the cytosensor microphysiometer and analyzed using inhibitors of signal transduction proteins. KEYWORDS: relaxin 3; GPCR135; relaxin receptor; cytosensor microphysi- ometer; relaxin; H3RLX INTRODUCTION GPCR135 1 until recently was an orphan G protein–coupled receptor, possessing sequence homology with the somatostatin and angiotensin II (AT1) receptors. How- ever, GPCR135 failed to bind to either somatostatin or angiotensin II and was cate- gorized as an orphan receptor and named SALPR (somatostatin and angiotensin-like peptide receptor). Recent studies have identified GPCR135 as the receptor for H3 re- laxin. 2 [ 125 I] H3 relaxin binds with high affinity to GPCR135, and unlabeled H3 re- laxin competes for this site. 2 Examination of other peptides in the relaxin family including porcine relaxin, insulin, INSL3, INSL4, INSL6, and H3 relaxin A-chain showed that they were unable to compete for [ 125 I] H3 relaxin binding. The only other ligand shown to compete for [ 125 I] H3 relaxin binding was H3 relaxin B-chain which competed with greater than 100-fold lower affinity than H3 relaxin. 2 Func- tionally, GPCR135 was shown to inhibit forskolin-induced cAMP accumulation suggesting that the receptor was coupled to G i , but no other signaling pathways have Address for correspondence: Prof. R. J. Summers, Department of Pharmacology, Monash Uni- versity, Wellington Road, Clayton 3800, Australia. Voice: +61-3-9905-1440; fax: +61-3-9905- 8192. roger.summers@med.monash.edu.au