We have recently reported that angiotensin II (Ang II)-induced mitogen-activated protein kinase (MAPK) activation is mainly mediated by Ca 2؉ -dependent activation of a protein tyrosine kinase through G q -coupled Ang II type 1 receptor in cultured rat vascular smooth muscle cells (VSMC). In the present study, we found Ang II rapidly induced the tyrosine phosphorylation of the epidermal growth factor (EGF) receptor and its association with Shc and Grb2. These reactions were inhibited by the EGF receptor kinase inhibitor, AG1478. The Ang II-induced phosphorylation of the EGF receptor was mimicked by a Ca 2؉ ionophore and completely inhibited by an intracellular Ca 2؉ chelator. Thus, AG1478 abolished the MAPK activation induced by Ang II, a Ca 2؉ ionophore as well as EGF but not by a phorbol ester or platelet-derived growth factor-BB in the VSMC. Moreover, Ang II induced association of EGF receptor with catalytically active c-Src. This reaction was not affected by AG1478. These data indicate that Ang II induces Ca 2؉ -dependent transactivation of the EGF receptor which serves as a scaffold for pre-activated c-Src and for downstream adaptors, leading to MAPK activation in VSMC.

Abstract-PYK2, a recently identified Ca 2ϩ -sensitive tyrosine kinase, has been implicated in extracellular signal-regulated kinase (ERK) activation via several G protein-coupled receptors. We have reported that angiotensin II (Ang II) induces Ca 2ϩ -dependent transactivation of the epidermal growth factor receptor (EGFR) which serves as a scaffold for preactivated c-Src and downstream adaptors (Shc/Grb2), leading to ERK activation in cultured rat vascular smooth muscle cells (VSMC). Herein we demonstrate the involvement of PYK2 in this cascade. Ang II rapidly induced tyrosine phosphorylation of PYK2, whose effect was completely inhibited by an AT 1 receptor antagonist and an intracellular Ca 2ϩ chelator. A Ca 2ϩ ionophore also induced PYK2 tyrosine phosphorylation to a level comparable with that by Ang II, whereas phorbol ester-induced phosphorylation was less than that by Ang II. Moreover, PYK2 formed a complex coprecipitable with catalytically active c-Src after Ang II stimulation. Although a selective EGFR kinase inhibitor completely abolished Ang II-induced recruitment of Grb2 to EGFR and markedly attenuated Ang II-induced ERK activation, it had no effect on Ang II-induced PYK2 tyrosine phosphorylation or its association with c-Src and Grb2. These data suggest that the AT 1 receptor uses Ca 2ϩ -dependent PYK2 to activate c-Src, thereby leading to EGFR transactivation, which preponderantly recruits Grb2 in rat VSMC.

We have studied whether activation of epidermal growth factor receptor (EGFR) is involved in stretch-induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation and protein synthesis in cultured rat vascular smooth muscle cells (VSMC). Cyclic stretch (1 Hz) induced a rapid (within 5 min) phosphorylation of ERK1/2, an effect that was time and strength dependent and inhibited by an EGFR kinase inhibitor (AG-1478) but not by a platelet-derived growth factor receptor kinase inhibitor (AG-1296). The stretch rapidly (within 2 min) induced tyrosine phosphorylation of several proteins, among which 180-kDa protein was shown to be EGFR as revealed by blockade with AG-1478 as well as immunoprecipitation with anti-EGFR antibody coupled with immunoblotting with anti-phosphotyrosine antibody. The stretch rapidly (within 2 min) induced association of tyrosine-phosphorylated EGFR with adaptor proteins (Shc/Grb2) as revealed by coprecipitation with glutathione-S-transferase-Grb2 fusion protein coupled with immunoblotting with anti-phosphotyrosine, anti-EGFR, and anti-Shc antibodies. Transfection of a dominant-negative mutant of H-Ras also inhibited stretch-induced ERK1/2 activation. Treatment with a stretch-activated ion channel blocker (Gd(3+)) and an intracellular Ca(2+) antagonist (TMB-8) inhibited stretch-induced phosphorylation of EGFR and ERK1/2. Treatment with AG-1478 and a mitogen-activated protein kinase kinase inhibitor (PD-98059), but not AG-1296, blocked [(3)H]leucine uptake stimulated by a high level of stretch. These data suggest that ERK1/2 activation by mechanical stretch requires Ca(2+)-sensitive EGFR activation mainly via stretch-activated ion channels, thereby leading to VSMC growth.

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Vascular smooth muscle cells (VSMC) from rat aorta possess specific receptors for a novel potent vasorelaxant peptide, adrenomedullin (AM). To elucidate its receptor coupling to guanine nucleotide-binding stimulatory protein and the structural requirement of the AM molecule to its vascular receptors, we have studied the effects of guanine nucleotides on [125I]human (h) AM binding and adenylate cyclase activity in cultured rat VSMC, and the effects of various synthetic hAM analogs on [125I]hAM binding and the cAMP response. Guanosine 5'-O-(3-thiotriphosphate) dose dependently inhibited [125I]hAM binding to rat VSMC membranes. hAM stimulated adenylate cyclase activity, and its effect was additive with GTP. hAM-induced cAMP formation was abrogated by pretreatment with cholera toxin, but not by that with pertussis toxin. Intact hAM-(1-52)-NH2 and N-terminal truncated derivatives [hAM-(13-52)-NH2, hAM-(16-52)-NH2] almost equally inhibited [125I]hAM binding and stimulated cAMP formation, whereas removal of C-terminal Tyr52 residue [hAM-(1-51)-NH2] remarkably decreased receptor-binding activity and the cAMP response. The effects of hAM-(1-52)-OH, hAM-(1-51)-OH, and a linear hAM analog ([carbamoylmethyl-Cys16,21]hAM-NH2) were far less potent on receptor binding and the cAMP response than that of hAM-(1-52)-NH2. The C-terminal fragment [hAM-(33-52)-NH2] and the N-terminal fragment [hAM-(1-10)-OH] had neither receptor-binding nor adenylate cyclase activity. hAM-(22-52)-NH2 had no agonistic effect, but showed an antagonistic effect on the hAM-induced cAMP response. These data suggest that vascular AM receptors are functionally coupled to adenylate cyclase via guanine nucleotide-binding stimulatory protein. Studies of the structure-activity relationship of hAM revealed that the cyclic structure formed by the disulfide bridge and amidation of the C-terminal residue of the AM molecule are critical for receptor binding and subsequent cAMP generation and suggest that the C-terminal fragment hAM-(22-52)-NH2 may be an antagonist for vascular AM receptors.

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