Activating Mutations of the Gsa Gene Are Associated with Low Levels of Gsa Protein in Growth Hormone- Secreting Tumors*

Evidence suggests the existence of a direct relationship between cellular Gsa content and activation of the adenylyl cyclase system. Data on Gsa levels in endocrine tumors that depend on cAMP for growth, particularly pituitary adenomas, are still limited. The levels of Gsa protein were evaluated in 11 GH-secreting adenomas with Gsa mutations (gsp) and 15 without (gsp). Complementary DNAs from gsp tumors contained very low amounts of wild-type Gsa sequences, indicating a preponderance of the mutant Gsa transcripts in these tumors. Immunoblotting of Gsa protein showed that the two isoforms were present at high levels in all gsp, but were undetectable or barely detectable in gsp. The low Gsa content in gsp 1 tumors was not due to a reduction in ribonucleic acid synthesis or stability, as Gsa messenger ribonucleic acid levels were similar in wild-type and mutant tissues. Treatment of gsp cells with cholera toxin caused a marked reduction of Gsa levels. As in other cell systems cholera toxin increases Gsa degradation, our data are consistent with an accelerated removal of mutant Gsa. This may represent an additional mechanism of feedback response to the constitutive activation of cAMP signaling in pituitary tumors with mutations in the Gsa gene. (J Clin Endocrinol Metab 83: 4386–4390, 1998) P adenomas are monoclonal neoplasia due to genetic alterations that cause abnormal growth either by activating growth stimulatory proteins or by inactivating growth inhibitory signals (1). Although in recent years a large number of protooncogenes and antioncogenes have been screened for mutations in pituitary tumors, only loss of heterozygosity in 11q13 and mutations in the gene encoding the a-subunit of the stimulatory G protein (Gsa) have reproducibly been found in these neoplasia (2, 3). Although mutations of the menin gene, recently localized in 11q13, probably have little role in pituitary tumorigenesis (4, 5), Gsa is a key element for the activation of the cAMP-dependent pathway that in pituitary cells is a signal for differentiation and proliferation (6). Point mutations that have been first identified in GH-secreting adenomas occur at two specific sites in the Gsa gene, codon 201 or 227, the common effect of both being to constitutively activate adenylyl cyclase by impairing the intrinsic guanosine triphosphatase activity of the subunit (2, 7). The same gain of function mutations have been subsequently identified in a subset of toxic thyroid adenomas and differentiated thyroid adenocarcinomas (8, 9), consistent with the hypothesis that in selected cell types Gsa gene may be converted into an oncogene, designated gsp for Gs protein (2, 7, 10–13). Expression of Gsa can vary over a wide range in human tissues, and several lines of evidence suggest the existence of a direct relationship between cellular Gsa content and activation of the adenylyl cyclase system (14). However, data on Gsa expression in endocrine tumors that depend on cAMP for growth are still limited and controversial. Increased levels of Gsa have been found in tumoral thyroid samples compared with those in normal tissue; the overexpression especially involves the mutant Gsa or is independent of the presence of mutations according to various reports (15–17). Whereas it has been recently suggested that high Gsa levels may be sufficient to stimulate phosphorylation of cAMP response element-binding protein in GH-secreting adenomas (18), the Gsa content in pituitary tumors with and without activating Gsa mutations has been poorly investigated to date. The present study shows extremely low amounts, if any, of Gsa in GH-secreting adenomas carrying Gsa mutations and investigates the molecular mechanisms underlying this phenomenon. Subjects and Methods