Date of Award

8-2003

Degree Type

Thesis

Degree Name

MS Biology

Department

Biology

Advisor

Allan D. Blake

Committee Member

Sulie L. Chang

Committee Member

Jane Ko

Keywords

Molecular pharmacolgy, Somatostatin receptors

Abstract

Somatostatin (somatotropin release inhibitory factor, SRIF), inhibits animal cell secretion and proliferation. SRIF is transcribed from a single gene as a 1 t 6 amino acid that in precursor is cleaved a cell specific manner into two distinct peptides, SRIF-14 and SRIF-28. Each binds with high affinity to all five known SRIF-receptor subtypes (sst, - ssts) and several of these receptor subtypes are endogenously expressed in the AtT-20 cell, a murine pituitary corticotroph. SRIF receptors belong to the G protein-coupled family of integral membrane receptors. Within the SRIF receptor family, it has been shown that the sst, receptor subtype exists as splice-variants (sstzA and sst28) that exhibit over-lapping tissue expression. The sstzA and sstze splice-variants occur within the carboxyl-tenninus of the receptor, an area of the protein, which is not involved in ligand binding, thereby making a phannacologic distinction of these two subtypes extremely difficult. Indeed, the function of these receptor splice-variants, as well as the significance of their cellular co-expression is unknown. To delineate the function of these closely related receptors, we have employed double-stranded RNA interference ( dsRNAi), a highly selective post-transcriptional gene silencing method. dsRNAi allows sst2A mRNA to be specifically targeted and inhibited while testing the functional capabilities of the remaining receptor population. When used in conjunction with receptor subtype selective agonists, dsRNAi provides a powerful genetic tool to explore receptor function. In the current study, we inhibit the expression of the sshA receptor with dsRNAi, and examine the functional consequences of this inhibition by monitoring receptor protein expression with subtype selective antibodies, as well as assessing the functional consequences of this inhibition on intracellular cAMP accumulation. Our results demonstrate that RNA silencing oligonucleotides against the sshA carboxyl terminus can be incorporated into AtT-20 cells in a time and concentration-dependent manner. Furthermore, the uptake of these sst2A inactivating oligonuclcotides, suppresses sst?A receptor expression and inhibits receptor cyclic nucleotide effects. Taken together, these results suggest that RNA interference is a promising approach to defining the function of highly homologous receptors that cannot be distinguished through conventional phannacologic approaches.

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