Abstract-In neonatal cardiomyocytes, activation of the G q -coupled ␣ 1 -adrenergic receptor (␣ 1 AR) induces hypertrophy by activating mitogen-activated protein kinases, including c-Jun NH 2 -terminal kinase (JNK). Here, we show that JNK activation is essential for ␣ 1 AR-induced hypertrophy, in that ␣ 1 AR-induced hypertrophic responses, such as reorganization of the actin cytoskeleton and increased protein synthesis, could be blocked by expressing the JNK-binding domain of JNK-interacting protein-1, a specific inhibitor of JNK. We also identified the classes and subunits of G proteins that mediate ␣ 1 AR-induced JNK activation and hypertrophic responses by generating several recombinant adenoviruses that express polypeptides capable of inhibiting the function of specific G-protein subunits. ␣ 1 AR-induced JNK activation was inhibited by the expression of carboxyl terminal regions of G␣ q , G␣ 12 , and G␣ 13 . JNK activation was also inhibited by the G␣ q/11 -or G␣ 12/13 -specific regulator of G-protein signaling (RGS) domains and by C3 toxin but was not affected by treatment with pertussis toxin or by expression of the carboxyl terminal region of G protein-coupled receptor kinase 2, a polypeptide that sequesters G␥. ␣ 1 AR-induced hypertrophic responses were inhibited by G␣ q/11 -and G␣ 12/13 -specific RGS domains, C3 toxin, and the carboxyl terminal region of G protein-coupled receptor kinase 2 but not by pertussis toxin. Activation of Rho was inhibited by carboxyl terminal regions of G␣ 12 and G␣ 13 but not by G␣ q . Our findings suggest that ␣ 1 AR-induced hypertrophic responses are mediated in part by a G␣ 12/13 -Rho-JNK pathway, in part by a G q/11 -JNK pathway that is Rho independent, and in part by a G␥ pathway that is JNK independent. Key Words: c-Jun NH 2 -terminal kinase Ⅲ G 12 family G proteins Ⅲ ␣ 1 -adrenergic receptors Ⅲ hypertrophy I n cardiomyocytes, stimulation of G-protein-coupled receptors activates mitogen-activated protein kinases, including c-Jun NH 2 -terminal kinase (JNK), which can subsequently trigger hypertrophic responses. 1 ␣ 1 -Adrenergic receptors (␣ 1 ARs) are G q -coupled receptors expressed in the heart, and they are thought to play a role in cardiac hypertrophy. Significant evidence points to an important role for G␣ q in receptor-induced hypertrophic responses. For example, ␣ 1 AR-induced hypertrophy can be inhibited by neutralizing antibodies that recognize G␣ q . In addition, a mutated M1 muscarinic acetylcholine receptor with impaired coupling to G q , in contrast to the wild-type receptor, is unable to induce hypertrophy. 2,3 It has also been reported that cardiac hypertrophy can be induced by expressing a constitutively active mutant of G␣ q . 4 In total, these results suggest that G␣ q plays an essential role in G q -coupled receptor-mediated hypertrophy, a conclusion strengthened by evidence that overexpression of protein kinase C, which is activated by diacylglycerol, can induce hypertrophy in a transgenic animal model. 5 Recent reports have demonstrated that var...
We examined the subtype-selective binding site of the beta-adrenergic receptors (betaARs). The beta(1)/beta(2)-chimeric receptors showed the importance of the second and seventh transmembrane domains (TM2 and TM7) of the beta(2)AR for the binding of the beta(2)-selective agonists such as formoterol and procaterol. Alanine-substituted mutants of TM7 of the beta(2)AR showed that Tyr(308,) located at the top of TM7, mainly contributed to beta(2) selectivity. However, Tyr(308) interacted with formoterol and procaterol in two different ways. The results of Ala- and Phe-substituted mutants indicated that the phenyl group of Tyr(308) interacted with the phenyl group in the N-substituent of formoterol (hydrophobic interaction), and the hydroxyl group of Tyr(308) interacted with the protonated amine of procaterol (hydrophilic interaction). In contrast to beta(2)AR, TM2 is a major determinant that beta(1)-selective agonists such as denopamine and T-0509 bound the beta(1)AR with high affinity. Three amino acids (Leu(110), Thr(117), and Val(120)) in TM2 of the beta(1)AR were identified as major determinants for beta(1)-selective binding of these agonists. Three-dimensional models built on the basis of the predicted structure of rhodopsin showed that Tyr(308) of the beta(2)AR covered the binding pocket formed by TM2 and TM7 from the upper side, and Thr(117) of the beta(1)AR located in the middle of the binding pocket to provide a hydrogen bonding for the beta(1)-selective agonists. These data indicate that TM2 and TM7 of the betaAR formed the binding pocket that binds the betaAR subtype-selective agonists with high affinity.
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