Agonist-induced Ca2+ Sensitization in Smooth Muscle: REDUNDANCY OF RHO GUANINE NUCLEOTIDE EXCHANGE FACTORS (RhoGEFs) AND RESPONSE KINETICS, A CAGED COMPOUND STUDY

Andra Stevenson,Zygmunt S Derewenda,Mykhaylo V Artamonov,Urszula Derewenda,J Silvio Gutkind,Avril V Somlyo,Ko Momotani,David R Trentham,Paul W Read

doi:10.1074/jbc.m113.514596

Abstract

Many agonists, acting through G-protein-coupled receptors and Gα subunits of the heterotrimeric G-proteins, induce contraction of smooth muscle through an increase of [Ca(2+)]i as well as activation of the RhoA/RhoA-activated kinase pathway that amplifies the contractile force, a phenomenon known as Ca(2+) sensitization. Gα12/13 subunits are known to activate the regulator of G-protein signaling-like family of guanine nucleotide exchange factors (RhoGEFs), which includes PDZ-RhoGEF (PRG) and leukemia-associated RhoGEF (LARG). However, their contributions to Ca(2+)-sensitized force are not well understood. Using permeabilized blood vessels from PRG(-/-) mice and a new method to silence LARG in organ-cultured blood vessels, we show that both RhoGEFs are activated by the physiologically and pathophysiologically important thromboxane A2 and endothelin-1 receptors. The co-activation is the result of direct and independent activation of both RhoGEFs as well as their co-recruitment due to heterodimerization. The isolated recombinant C-terminal domain of PRG, which is responsible for heterodimerization with LARG, strongly inhibited Ca(2+)-sensitized force. We used photolysis of caged phenylephrine, caged guanosine 5'-O-(thiotriphosphate) (GTPγS) in solution, and caged GTPγS or caged GTP loaded on the RhoA·RhoGDI complex to show that the recruitment and activation of RhoGEFs is the cause of a significant time lag between the initial Ca(2+) transient and phasic force components and the onset of Ca(2+)-sensitized force.

Highlights

Tissues deficient in either PRG or leukemia-associated RhoGEF (LARG) showed significantly delayed onset of ET-1-induced Ca2ϩ-sensitized force and required a much longer time to reach maximum contraction. We explored this further by directly measuring times to activation using photolysis of caged phenylephrine, caged guanosine 5؅-O-(thiotriphosphate) (GTP␥S) in the bath solution, and caged GTP␥S or caged GTP loaded on the RhoA1⁄7RhoGDI complex or on the G14V RhoA1⁄7RhoGDI complex, respectively, to show that the recruitment and activation of RhoGEFs is the cause of a significant time lag between the initial Ca2ϩ transient and phasic force components and the onset of Ca2ϩ-sensitized force
LARG, p115RhoGEF, and p63RhoGEF protein expression levels were similar in both animals as were those for G␣q/11, G␣12, G␣13, and thromboxane A2 (TXA2) and ET-1 receptors (Fig. 3, B and C)
Photolytic release of saturating concentrations of phenylephrine (2 ␮M) displayed much slower kinetics than the GTP␥S-RhoA1⁄7RhoGDI complex with a long delay (16 s) having a latent phase with no measurable force followed by a lag phase

Summary

Introduction

Conclusion: Ca2ϩ-sensitized force is induced by parallel signaling through RhoGEFs, which are rate-limiting due to their slow recruitment and activation. Many agonists, acting through G-protein-coupled receptors and G␣ subunits of the heterotrimeric G-proteins, induce contraction of smooth muscle through an increase of [Ca2؉]i as well as activation of the RhoA/RhoA-activated kinase pathway that amplifies the contractile force, a phenomenon known as Ca2؉ sensitization. G␣12/13 subunits are known to activate the regulator of G-protein signaling-like family of guanine nucleotide exchange factors (RhoGEFs), which includes PDZ-RhoGEF (PRG) and leukemia-associated RhoGEF (LARG). Their contributions to Ca2؉-sensitized force are not well understood. The isolated recombinant C-terminal domain of PRG, which is responsible for heterodimerization with LARG, strongly inhibited Ca2؉-

Methods

Results

Conclusion