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Human somatostatin receptor subtypes (SSTR1‐5) bind their natural ligands SRIF‐14 and SRIF‐28 with high affinity. By contrast, short synthetic SRIF analogues such as SMS 201‐995, a peptide agonist used for the treatment of various endocrine and malignant disorders, display sub‐nanomolar affinity only for the receptor subtype SSTR2. To understand the molecular nature of selective peptide agonist binding to somatostatin receptors we have now, by site‐directed mutagenesis, identified amino acids mediating SMS 201‐995 specificity for SSTR2. Sequentially, amino acids in SSTR1, a receptor subtype exhibiting low affinity for SMS 201‐995, were exchanged for the corresponding SSTR2 residues. After three consecutive steps, in which eight amino acids were exchanged, a SSTR1 mutant receptor with high affinity for SMS 201‐995 was obtained. Receptor mutants with different combinations of these eight amino acids were then constructed. A single Ser305 to Phe mutation in TM VII increased the affinity of SSTR1 for SMS 201‐995 nearly 100‐fold. When this mutation was combined with an exchange of Gln291 to Asn in TM VI, almost full susceptibility to SMS 201‐995 was obtained. Thus, it is concluded that the specificity of SMS 201‐995 for SSTR2 is mainly defined by these two amino acids in transmembrane domains VI and VII. Using the conjugate gradient method we have, by analogy to the well established structure of bacteriorhodopsin, built a model for SRIF receptor‐ligand interactions that explains the importance of Gln291 and Ser305 for the selectivity of agonists.

A number of substituted indole carbazimidamides were prepared and evaluated as 5-HT4 receptor agonists by using the isolated field-stimulated guinea pig ileum preparation. Their selectivity for the 5-HT4 receptor was established by examining their affinity for other 5-HT receptors using radioligand-binding techniques. Several selective and highly potent full as well as partial agonists emerged from this study. For example, 1b,d were found to be the most potent, full 5-HT4 receptor agonist described so far (EC50 = 0.5 and 0.8 nM, respectively), being 6 and 4 times more potent than serotonin itself. On the other hand, 5b and 1h appeared as partial 5-HT4 receptor agonists in the nonstimulated guinea pig ileum preparation with potencies, evaluated against serotonin action, respectively similar (5b, Ki = 12 nM) to and 300-fold higher (1h, Ki = 0.04 nM) than serotonin.

To identify determinants that form nonapeptide hormone binding domains of the white sucker Catostomus commersoni [Arg8] vasotocin receptor, chimeric constructs encoding parts of the vasotocin receptor and parts of the isotocin receptor have been analyzed by [(3,5-3H) MATERIALS AND METHODSMaterials. [(3, 5-3H)Tyr2, Arg8]vasotocin (specific activity 16 Ci/mmol; 1 Ci = 37 GBq) was purchased from DuPont.Construction of Mutants. To exchange parts of the VTR and the ITR, restriction sites were introduced into appropriate positions of the VTR or ITR cDNAs; these were located as indicated in Fig. 1. Individual cDNA fragments were amplified by using a thermophilic DNA polymerase (Ventpolymerase; New England Biolabs) using primers specific for either the 5' or 3' parts of the nonapeptide receptor cDNAs and internal mutagenic primers. Amplified DNA fragments that overlapped at the introduced restriction sites were cut with the appropriate endonuclease, ligated, and cloned into either pBluescript or pcDNA3. Functional Expression Assays in Oocytes. Plasmids, which were linearized in the polylinker region, served as templates for the synthesis of in vitro transcribed cRNAs by using a T7 RNA polymerase transcription kit (Promega). Injection of cRNAs into frog oocytes and voltage-clamp measurements were performed as described (7).Molecular Modeling. The sequences of the VTR and ITR and the rat Via receptor were aligned with those of the somatostatin and serotonin receptors as well as with that of bacteriorhodopsin. The precise locations of the first and last amino acids of the TMs were determined by comparison with