Thyroid hormone receptor-induced bending of specific DNA sequences is modified by an accessory factor.

Transcriptional regulation by thyroid and steroid hormone receptors requires their recognition and binding of specific DNA sequences. However, little is known about the mechanisms whereby DNA bound receptors regulate transcription. In the present study, we examined the effects of thyroid hormone receptor (TR) binding on DNA conformation using various TR recognition sites contained within sets of circularly permuted flanking sequences. We show that under conditions where TR binds predominantly as monomer, the conformation of a number of binding sites is changed in a manner consistent with receptor induced bending. Despite similar affinities for receptor binding, not all binding sites tested showed evidence for receptor-induced bending. Notably, the conformation of a sequence from the frog vitellogenin 2 gene, which confers a positive transcriptional response when bound by estrogen receptor (ER), but a negative response when bound by TR, appeared to be unaffected by binding of either TR or ER. The observations suggest that the ability of the receptor to alter DNA architecture is strongly dependent on sequence characteristics other than those required for receptor binding. While both partly purified TR from rat liver and TR translated in vitro were able to induce DNA bending, the bend centers and bend angles produced by these different sources of receptor differed. However, addition of a receptor-depleted fraction from the rat liver TR preparation to in vitro translated receptor stimulated TR binding and appeared to form heterodimers with TR. This resulted in changes in both bend centers and bend angles to resemble more closely those produced by native receptor. Together, these results suggest that receptor-induced DNA bending may be specific to TRs and that the position and degree of bending is further modulated by the formation of heterodimers between TRs and accessory protein(s).

The angiotensin AT2 receptor stimulates protein tyrosine phosphatase activity and mediates inhibition of particulate guanylate cyclase.

The signalling mechanism and cellular targets of the AT2 receptor are still unknown. We report that angiotensin II (Ang II) inhibits basal and atrial natriuretic peptide stimulated particulate guanylate cyclase (pGC) activity through AT2 receptors in rat adrenal glomerulosa and PC12W cells. This inhibition is blocked by the phosphotyrosine phosphatase (PTPase) inhibitor orthovanadate but not by the Ser/Thr phosphatase inhibitor okadaic acid, suggesting the involvement of a PTPase in this process. Moreover, Ang II induces a rapid, transient and orthovanadate sensitive dephosphorylation of phosphotyrosine containing proteins in PC12W cells. Our findings suggest that AT2 receptors signal through stimulation of a PTPase and that this mechanism is implicated in the regulation of pGC activity. This observation is also the first example of hormonal inhibition of basal pGC activity.

Angiotensin II AT2 receptors do not interact with guanine nucleotide binding proteins.

We have studied the effect of GTP gamma S on the affinity and binding kinetics of angiotensin II in plasma membrane particulate prepared from tissues expressing either only AT1 (human renal artery smooth muscle cells), only AT2 (human myometrium and bovine cerebellar cortex) or both angiotensin II receptor subtypes (rat adrenal glomerulosa). We also examined the ability of angiotensin II to stimulate GTP gamma[35S] incorporation in these membrane preparations. In contrast to its effects on angiotensin II binding to the AT1 receptor, GTP gamma S does not affect binding parameters to the AT2 receptor. Moreover, in tissues expressing solely AT2 receptors, angiotensin II was unable to induce GTP gamma[35S] incorporation. These findings indicate that AT2 receptors do not interact with G-proteins and that angiotensin II must therefore mediate some of its effects through G-protein-independent mechanisms.