Expression of functional stimulatory guanine nucleotide binding protein in nonfunctioning thyroid adenomas is not correlated to adenylate cyclase activity and growth of these tumors.

Abstract

In thyroid cells, the alpha-subunit of the stimulatory guanine nucleotide binding protein (Gs alpha) acts as signal transducer between the TSH receptor and the adenylate cyclase (AC), and it regulates both growth and function. In order to analyze Gs alpha expression by both Western blot analysis and in situ, we generated an antibody raised against a recombinant human Gs alpha protein. With this antibody, a strong cytoplasmic Gs alpha-immunostaining was detectable in cultured human thyroid cells and in TSH-stimulated rat thyroids, in contrast to normal human thyroids and to T4-treated rat thyroids, which showed only weak immunoreactivity. We obtained the following results by immunohistochemistry and Western blot analysis of 32 actively growing human thyroid adenomas: 1) strong Gs alpha expression in 11 adenomas, including 4 hyperfunctioning nodules, 1 of these with a point mutation in codon 201 of the Gs alpha gene; 2) no expression or only weak Gs alpha expression in 13 adenomas; and 3) a pattern of Gs alpha-positive and Gs alpha-negative cells in the remaining 8 adenomas. In addition, we analyzed ADP-ribosylation of Gs alpha and AC activity in 9 nonfunctioning adenomas and found a significant correlation between Gs alpha immunoreactivity and ADP-ribosylation and no correlation of both with basal and TSH-stimulated AC activity. In both types of adenomas, i.e. those with high as well as low Gs alpha, an indistinguishably high fraction of proliferating cells was detectable. We conclude that expression of functional Gs alpha protein in nonfunctioning thyroid adenomas is neither correlated to the basal or TSH-stimulated AC activity nor to the proliferation rate of these tumors.

Expression of G(alpha)(s) proteins and TSH receptor signalling in hyperfunctioning thyroid nodules with TSH receptor mutations

“…However, the mean Gai and Gaq/11 protein expression in our HTNs and CTNs as well as the Gas protein expression in our CTNs was not different compared with their surrounding tissue. Similar results have previously been reported for CTNs (8). Therefore, overexpression of Gas, Gai and Gaq/11 subunits is unlikely to mediate the increased mitogenic signalling in HTNs or CTNs.…”

“…Our results of decreased expression of Gas in HTNs with TSHR mutations are difficult to reconcile with this report by Derwahl and co-workers. We and others (8) have shown that both Ga protein and cAMP content in thyroid tumours varies greatly. However, the HTNs included in our study were screened for TSH receptor mutations and mutations in the Gas protein gene by DNA sequencing and DGGE (12,16,24) and were also examined for their clonality (25).…”

“…Reduced expression of Gas proteins in HTNs (9), increased Gas protein expression (7,8) or no change in Gas protein expression (10) in HTNs have recently been reported. The samples examined in former studies of Gas protein expression, except those described by Derwahl et al (7), were not analysed for mutations in the TSHR or in the Gas protein (8,10,22,23) or have only been screened for Gas protein mutations (22). Some investigators used, in addition to Western blotting, ADP ribosylation experiments to investigate G protein expression (7)(8)(9).…”

“…The samples examined in former studies of Gas protein expression, except those described by Derwahl et al (7), were not analysed for mutations in the TSHR or in the Gas protein (8,10,22,23) or have only been screened for Gas protein mutations (22). Some investigators used, in addition to Western blotting, ADP ribosylation experiments to investigate G protein expression (7)(8)(9). A further aspect reducing the strength of the former results is the low number of HTNs investigated, 2 (23), 7 (9), 4 (8), 10 (7) and 9 (10) respectively.…”

“…Moreover, we also determined the expression of Ga subunits in HTNs with different molecular aetiologies and in cold thyroid nodules (CTNs) which also show increased proliferation (8) together with decreased thyroid hormone production.…”