Molecular cloning and functional characterization of a rat somatostatin sst_2(b) receptor splice variant

Abstract

1. The mouse somatostatin (SRIF) sst₂ receptor exists in two splice variants, sst_2(a) and sst_2(b), which differ in their intracellular carboxy-termini only. The murine sst_2(b) receptor was reported to be less prone to agonist-induced desensitization as compared with the sst_2(a) receptor. To determine whether a sst_2(b) splice variant with similar functional characteristics exists in the rat, we have isolated a cDNA fragment from rat gastric mucosa encoding a sst_2(b) receptor and expressed the full-length protein in CHO-K1 cells for functional characterization.

2. This study provides the first evidence for the occurrence in the rat of the sst_2(b) receptor, which has a 15 amino acid carboxy-terminus differing in composition to the 38 amino acid C-terminus of the rat sst_2(a) receptor.

3. In CHO-K1 cells expressing rat recombinant sst_2(a) or sst_2(b) receptors, SRIF caused concentration-dependent increases in extracellular acidification rates (EAR) with pEC₅₀ values of 9.0 and 9.9, respectively. Pre-treatment with pertussis toxin (Ptx) caused a rightward displacement of the SRIF concentration-effect curves with pEC₅₀ values of 8.3 (sst_2(a)) and 8.4 (sst_2(b)).

4. SRIF (3 pM–3 nM) also caused concentration-dependent inhibition of forskolin-stimulated cyclic AMP formation in CHO-sst_2(a) cells (pIC₅₀ 10.5) and CHO-sst_2(b) cells (pIC₅₀ 10.4). The degree of inhibition was less with higher concentrations of SRIF resulting in bell-shaped concentration-effect curves. Following pre-treatment with Ptx, the inhibitory effect of SRIF was abolished and SRIF caused only increases in cyclic AMP formation.

5. Both the SRIF-induced increases in EAR and inhibition of cyclic AMP formation were susceptible to agonist-induced desensitization, but this was less apparent following pre-treatment with Ptx.

6. This demonstrates that the operational characteristics of the recombinant rat sst_2(a) and sst_2(b) receptors are broadly similar. Both isoforms couple to Ptx-sensitive as well as -insensitive G proteins and are equally prone to agonist-induced desensitization.

Full Text

The Full Text of this article is available as a PDF (416.5 KB).