Molecular and functional properties of somatostain receptor subtypes.

Identification of the ligand binding domains of the somatostain (SRIF) receptors may facilitate the rational development of new SRIF ligands. To identify ligand-binding domains of sst1, and sst2, we tested a series of chimeras. Using site-directed mutagenesis, we found that to bind with high affinity to sst2, the sst2 agonists MK678 and SMS-201-995 require a four amino acid sequence (FDFV) at the border of the third extracellular loop and transmembrane 7. Transference of residue 294 in msst2 to sst1 conferred onto sst1 the ability to bind SMS-201-995 and other octapeptides. Cyclic peptides with a phenylalanine adjacent to the D-Trp appear to interact with Phe294 of sst2, whereas hexapeptides with a tyrosine adjacent to the D-Trp, such as MK 678 and BIM 23027, did not interact with the Phe294. We have recently identified a peptide that selectively binds to human (h)sst1, with 100-fold higher affinity than for the other cloned SRIF receptor subtypes. The second extracellular loop of sst1 is critical for this peptide to bind. This contrasts with the sites involved in binding of sst2 agonists and indicates that the two receptors have distinct ligand-binding domains. G proteins couple SRIF receptors to multiple cellular effector systems, including adenylyl cyclase and ionic conductance channels. A critical cellular action of SRIF is the inhibition of Ca2+ influx, which may be responsible for its blockade of hormone and neurotransmitter release. Various studies suggest that both sst2 and sst5 endogenously expressed in AtT-20 cells can couple to L-type Ca2+ channels; the coupling was pertussis toxin-sensitive. The coupling of sst2 to the Ca2+ channels was relatively resistant to desensitisation; 5 hours of pretreatment with MK 678 did not attenuate MK 678 inhibition of the Ca2+ current. In contrast, the sst5 receptors were desensitised by 1 hour of pretreatment with BIM 23052. Thus, the coupling of the two receptors to the Ca2+ channel could be differentially regulated. The SRIF receptor subtype coupling to the Ca2+ channel could also be distinguished by a unique antagonist, the peptide L362,855, which binds with high affinity to cloned sst5.