Abstract
The pattern of expression of somatostatin receptor (SSTR) genes and gene products in AtT-20 cells was characterized in an attempt to explain the SST-28 binding selectivity that typifies these cells. AtT-20 cells expressed multiple SSTR mRNAs. Paradoxically, this included mRNA for three of the four SST-14 selective receptors: SSTR2 ( +), SSTR1 (+), SSTR4 (+). The SST-28 selective SSTR5 was expressed as a 3.8-kilobase (kb) transcript of relatively low abundance (+) in contrast to normal mouse pituitary which displayed high levels ( ) of a 2.4-kb SSTR5 mRNA. Immunoblot analysis of solubilized membranes with an antipeptide SSTR2 antibody revealed a single SSTR2 protein of 72 +/- 2 kDa. Preincubation of AtT-20 cell membranes with SSTR2 antibody reduced 125I-[Leu8,D-Trp22,Tyr25]SST-28 binding sites by 38%. Residual binding sites exhibited a 4.9-fold increase in affinity for SST-28, a 2.6-fold decrease in affinity for SST-14, and an SST-28:SST-14 potency ratio of 40:1 compared with a potency ratio of 3.5:1 in control membranes. These results demonstrate the expression of four SSTR genes in AtT-20 cells of which SSTR2 predominates. Blockade of SSTR2 with antibody exposes high affinity SST-28 selective sites with comparable binding characteristics to those reported for cloned SSTR5. These SST-28 binding sites may arise from a SSTR5 variant encoded by a high molecular weight 3.8-kb transcript or more likely from another as yet undiscovered member of the SST-28 selective SSTR subfamily.
Highlights
Thepatternof expression of somatostatin receptor ceptor (SSTR) is heterogeneous, in part due to thperesence of (SSTR)genes and gene products in AtT-20 cells was char- subtypes selective for SST-14 and SST-28 [5, 6, 9, 12, 13].Reacterized in an attempt to explain the SST-28 binding ceptors t h a t show preference for SST-14 binding occur in cereselectivity that typifies these cells
Referred to as SSTR5)2 cloned in the rat and human exhibits 15-30-fold greater affinity for SST-28 than SST-14 and is truly SST-28-selective (24h3 We have reported recently t h a t AtT-20 cells express high levels of mRNA for SSTR2, a SST-14-selective receptor, plasma membranes prepared from these cells display overall SST-28 binding [32]
This suggests that either the SSTR2 mRNA is not expressed as a functional receptor protein or that thesecells feature higherexpression of a Somatostatin (SST)’ exists as two naturally occurring pep- SST-28-selective SSTR subtype(s) such as SSTR5
Summary
Thepatternof expression of somatostatin receptor ceptor (SSTR) is heterogeneous, in part due to thperesence of (SSTR)genes and gene products in AtT-20 cells was char- subtypes selective for SST-14 and SST-28 [5, 6, 9, 12, 13].Reacterized in an attempt to explain the SST-28 binding ceptors t h a t show preference for SST-14 binding occur in cereselectivity that typifies these cells. Referred to as SSTR5)2 cloned in the rat and human exhibits 15-30-fold greater affinity for SST-28 than SST-14 and is truly SST-28-selective The antibody displayed dilution-dependent inhibition of '251-[LTl'lSST-28 binding to brain and AtT-20 cell membrane receptors
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