Ligand Binding Pocket of the Human Somatostatin Receptor 5: Mutational Analysis of the Extracellular Domains
- Michael T. Greenwood1,
- Nedim Hukovic1,
- Ujendra Kumar1,
- Rosemarie Panetta1,
- Siv A. Hjorth2,
- Coimbatore B. Srikant1 and
- Yogesh C. Patel1
- 1Fraser Laboratories, Departments of Medicine and Neurology and Neurosurgery, McGill University, Royal Victoria Hospital, and the Montreal Neurological Institute, Montreal, Quebec H3A 1A1, Canada (M.T.G., N.H., U.K., R.P., C.B.S., Y.C.P.), and 2Laboratory for Molecular Pharmacology, The Laboratory Center, Rigshospitalet, Copenhagen, DK-2100, Denmark (S.A.H.)
Abstract
The ligand binding domain of G protein-coupled receptors for peptide ligands consists of a pocket formed by extracellular and transmembrane domain (TM) residues. In the case of somatostatin (SRIF), however, previous studies have suggested that the binding cavity of the octapeptide analog SMS201–995 (SMS) is lined by residues in TMs III–VII. The additional involvement of the extracellular domains for binding SMS or the natural SRIF ligands (SRIF-14, SRIF-28) has not been clarified. Using a cassette construct cDNA for the human somatostatin 5 receptor (sst5R), we systematically examined the role of exofacial structures in ligand binding by creating a series of mutants in which the extracellular portions have been altered by conservative segment exchange (CSE) mutagenesis for the extracellular loops (ECLs) and by deletion (for the NH2-terminal segment) or truncation analysis (ECL3). CHO-K1 cells were stably transfected with wild type or mutant human sst5R constructs, and agonist binding was assessed using membrane binding assays with125I-LTT SRIF-28 ligand. Deletion of the NH2terminus or CSE mutagenesis of ECL1 and ECL3 produced minor 2–8-fold decreases in affinity for SRIF-14, SRIF-28, and SMS ligands. Truncation of ECL3 to mimic the size of this loop in sst1R and sst4R (the two subtypes that do not bind SMS) did not interfere with the binding of SMS, SRIF-14, or SRIF-28. In contrast, both ECL2 mutants failed to bind 125I-LTT SRIF-28. Immunocytochemical analysis of nonpermeabilized cells with a human sst5R antibody revealed that the mutant receptors were targeted to the plasma membrane. Labeled SMS (125I-Tyr3 SMS) also failed to bind to the mutant ECL2 receptors. These results suggest a potential contribution of ECL2 (in addition to the previously identified residues in TMs III–VII) to the SRIF ligand binding pocket.
Footnotes
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Send reprint requests to: Dr. Y. C. Patel, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec H3A 1A1, Canada. E-mail: patel{at}rvhmed.lan.mcgill.ca
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This work was supported by Grants MT 10411 and MT 12603 from the Canadian Medical Research Council and Grant NS32160 from the National Institutes of Health. M.T.G. and N.H. contributed equally. Both are Fellows of the Fonds de la Recherche en Santé du Quebec. Y.C.P. is a Distinguished Scientist of the Canadian Medical Research Council.
- Abbreviations:
- GPCR
- G protein-coupled receptor
- ECL
- extracellular loop
- TM
- transmembrane domain
- CHO
- Chinese hamster ovary
- SRIF
- somatostatin
- SMS
- SMS201–995
- CSE
- conservative segment exchange
- sstR
- somatostatin receptor
- hsst5R
- human somatostatin 5 receptor
- sstxR
- somatostatin receptor, where x is the number of the receptor
- PCR
- polymerase chain reaction
- PBS
- phosphate-buffered saline
- BSA
- bovine serum albumin
- AT
- angiotensin
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
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- Received November 15, 1996.
- Accepted July 30, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
