Cloning and expression of a rat Smad1: Regulation by TGFβ and modulation by the Ras/MEK pathway

A new family of signaling intermediates for TGFβ superfamily members and other growth factors has recently been identified and termed Smads. It has been suggested that the Smad1 subfamily is regulated primarily by the TGFβ superfamily member bone morphogenetic protein (BMP). Here we demonstrate that TGFβ induced phosphorylation of endogenous Smad1 in untransformed IECs and that the RI and RII TGFβ receptors were detectable in Smad1 immunocomplexes. Expression of a dominant-negative mutant of Ras inhibited the ability of TGFβ to phosphorylate endogenous Smad1. In a separate series of experiments, we have cloned a rat homologue of the drosophila mad gene (termed RSmad1) by screening an intestinal epithelial cell (IEC) cDNA library. By using an in vitro kinase assay with RSmad1 as the substrate, we demonstrate that the TGFβ receptor complex can directly phosphorylate RSmad1. We show, further, that a dominant-negative mutant of MEK1 inhibited the ability of RSmad1 to induce the TGFβ-responsive reporter p3TP-Lux in a human breast cancer cell line. Collectively, our data demonstrate that TGFβ can regulate Smad1 and that the Ras and MEK signaling components are partially required for the ability of TGFβ to regulate Smad1.link_to_subscribed_fulltex

Structural basis of beta-adrenergic receptor subtype specificity studied with chimeric beta 1/beta 2-adrenergic receptors.

The beta 1- and beta 2-adrenergic receptors are two structurally related, but pharmacologically distinguishable, receptor subtypes, both of which activate adenylyl cyclase in a catecholamine-dependent manner through the guanine nucleotide-binding regulatory protein Gs. The receptors are approximately 50% identical in amino acid sequence and each is characterized by the presence of seven putative transmembrane domains. To elucidate the structural basis for the pharmacological distinctions between these two receptor subtypes, we constructed a series of chimeric beta 1/beta 2-adrenergic receptor genes and expressed them by injection of RNA into Xenopus laevis oocytes. The pharmacological properties of the expressed chimeric receptor proteins were assessed by radioligand binding and adenylyl cyclase assays utilizing subtype-selective agonists and antagonists. Our data indicate that transmembrane region IV is largely responsible for determining beta 1 vs. beta 2 properties with respect to agonist binding (relative affinities for epinephrine and norepinephrine). Transmembrane regions VI and VII play an important role in determining binding of beta 1 vs. beta 2 selective antagonists. However, a number of the other transmembrane regions also contribute, to a lesser extent, to the determination of beta-adrenergic receptor subtype specificity for agonists and antagonists. Thus, several of the membrane-spanning regions appear to be involved in the determination of receptor subtype specificity, presumably by formation of a ligand-binding pocket, with determinants for agonist and antagonist binding being distinguishable