BIOCHEMICAL PROPERTIES OF SOMATOSTATIN RECEPTORS

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Somatostatin 14 (SRIF) is a neurotransmitter in the central nervous system. It has been shown to have a number of functions in the brain including regulating neuronal firing and neurotransmitter release. The peptide may also play a role in the expression of certain behaviors such as cognition. Abnormalities of SRIF transmission in the brain have been implicated in a variety of mental disorders such as Schizophrenia, depression and Alzheimer's disease. Despite the importance of SRIF as a neurotransmitter, little is known about how this peptide induces it's physiological effects. The objective of this proposal is to better understand the molecular mechanisms of action of SRIF in brain. This will be accomplished by identifying the physical properties of the SRIF receptor. By identifying the properties of the SRIF receptor we may gain insight into how the receptor interacts with different transducing systems such as the adenylate cyclase system and ionic conductance channels. To investigate the biochemical properties of the SRIF receptor, we will purify the rat brain SRIF receptor using affinity chromatographic techniques. To demonstrate that the purified protein is the SRIF receptor, we will attempt to specifically label the protein with the SRIF analogue [125I] CGP 23996. Attempts will also be made to reconstitute the ability of the purified receptor to mediate SRIF stimulation of GTPase activity or SRIF inhibition of adenylate cyclase activity in phospholipid vesicles or cells deficient in the SRIF receptor. Once the purified protein is established to be the SRIF receptor, some of the physical properties of the receptor will be assessed. The size and charge of the receptor will be determined using gel electrophoretic techniques. The oligosaccharide content of the receptor will be investigated using both glycolytic enzymes and lectin chromatography. To further characterize the properties of the SRIF receptor, antibodies will be generated against the receptor. To generate the antibodies, the purified SRIF receptor will be partially sequenced using a gas phase microsequenator. The sequence information will be used to synthesize peptide fragments of the receptor. Polyclonal antibodies will be generated against these peptides. If antibodies that selectively interact with the SRIF receptor are developed, we will use these antibodies to further examine the biochemical properties of brain SRIF receptors as well as to determine whether there are physical differences between subtypes of SRIF receptors. There is functional evidence that SRIF receptor subtypes exist in brain with differing affinities for SRIF and its prohormone somatostatin 28. Demonstration that these receptor subtypes are physically distinct would support the notion that SRIF and somatostatin 28 are distinct neurotransmitters. Differences in the structure of somatostatin 28 and SRIF receptors will be investigated in two cell lines which exclusively express one receptor subtype or the other. The size, charge, peptide maps, sugar content and processing events which these receptor subtypes undergo will be examined and compared.