Date of Award

7-1994

Degree Type

Dissertation

University or Center

Clark Atlanta University(CAU)

School

School of Arts and Sciences

Degree Name

Ph.D.

Department

Biology

Abstract

The mas proto-oncogene encodes a seven transmembrane protein (MAS) which is suggested to function as a receptor for angiotensin. It (MAS) was initially identified in NIH-3T3 cells that were transformed with DNA isolated from a human epidermoid carcinoma. These cells formed foci in culture and tumors when injected into nude mice. On the other hand, untransformed cells did not. Further analysis of these cells showed that transformed cells bind increased levels of angiotensin when compared to untransformed cells. These studies also demonstrated that the Mas protein was structurally similar to the angiotensin receptor transmembrane proteins, AT1 and AT2 . This investigation was undertaken to examine the ability of the Mas protein to function as an receptor for angiotensin and promote cell proliferation. To this end, quantitation of mas genes by Polymerase Chain Reaction (PCR) and serial dilutions, and Southern blot analysis support an increased in mas genes in transformed cells. Northern blot analysis demonstrated an increased expression of the mas gene in transformed cells. No changes in the level of the AT2 angiotensin receptor gene expression was observed in the transformed and untransformed cell lines. Expression of the AT1 angiotensin receptor gene was not observed in these cell lines. Anti-peptide antibodies were generated against the 1st and 2nd extracellular regions of the Mas protein. Flow cytometric analysis using these antibodies indicated an increased presence of the Mas protein on the surf ace of transformed cells recognized by anti-peptide antibodies.

Western blot analysis showed two cross-reacting proteins of approximately ll0kd and 66kd in transformed cells; whereas, only a 66kd protein was found in untransformed cells.

Transformed cells exposed to mas antisense oligos greatly reduced the synthesis of Mas, decreased cell proliferation and the binding of angiotensin. Binding studies using [3H]-DUP-

753 (a non-peptidyl ligand which recognizes Ang subtype AT1 receptors) showed little binding to transformed cells.

Similar studies using PD-123319 (a non-peptidyl ligand that recognizes AT2 subtype receptors) indicated that approximately 60% of [125I]-Ang II was displaced using PD-123319. Further binding analysis of transformed cells suggests that [Sarl]-Ang II (an Ang II antagonist) could not completely displace [ 125I]-Ang II. Taken together, these data suggest that Mas protein is an Ang receptor which functions in the regulation of cell proliferation. Mas appears to be a member of a subtype different from AT1 or AT2.

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Biology Commons

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