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G-protein-coupled receptor kinase interacting protein-1 is required for pulmonary vascular development.

ABSTRACT: The G-protein-coupled receptor kinase interacting protein-1 (GIT1) is a multidomain scaffold protein that participates in many cellular functions including receptor internalization, focal adhesion remodeling, and signaling by both G-protein-coupled receptors and tyrosine kinase receptors. However, there have been no in vivo studies of GIT1 function to date.To determine essential functions of GIT1 in vivo, we generated a traditional GIT1 knockout mouse. GIT1 knockout mice exhibited approximately 60% perinatal mortality. Pathological examination showed that the major abnormality in GIT1 knockout mice was impaired lung development characterized by markedly reduced numbers of pulmonary blood vessels and increased alveolar spaces. Given that vascular endothelial growth factor (VEGF) is essential for pulmonary vascular development, we investigated the role of GIT1 in VEGF signaling in the lung and cultured endothelial cells. Because activation of phospholipase-Cgamma (PLCgamma) and extracellular signal-regulated kinases 1/2 (ERK1/2) by angiotensin II requires GIT1, we hypothesized that GIT1 mediates VEGF-dependent pulmonary angiogenesis by modulating PLCgamma and ERK1/2 activity in endothelial cells. In cultured endothelial cells, knockdown of GIT1 decreased VEGF-mediated phosphorylation of PLCgamma and ERK1/2. PLCgamma and ERK1/2 activity in lungs from GIT1 knockout mice was reduced postnatally.Our data support a critical role for GIT1 in pulmonary vascular development by regulating VEGF-induced PLCgamma and ERK1/2 activation.

SUBMITTER: Pang J

PROVIDER: S-EPMC2732662 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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G-protein-coupled receptor kinase interacting protein-1 is required for pulmonary vascular development.

Pang Jinjiang J Hoefen Ryan R Pryhuber Gloria S GS Wang Jing J Yin Guoyong G White R James RJ Xu Xiangbin X O'Dell Michael R MR Mohan Amy A Michaloski Heidi H Massett Michael P MP Yan Chen C Berk Bradford C BC

Circulation 20090309 11

<h4>Background</h4>The G-protein-coupled receptor kinase interacting protein-1 (GIT1) is a multidomain scaffold protein that participates in many cellular functions including receptor internalization, focal adhesion remodeling, and signaling by both G-protein-coupled receptors and tyrosine kinase receptors. However, there have been no in vivo studies of GIT1 function to date.<h4>Methods and results</h4>To determine essential functions of GIT1 in vivo, we generated a traditional GIT1 knockout mou ...[more]

PMID: 19273721

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Project description:Significance statementG protein-coupled receptor kinase 4 (GRK4) regulates renal sodium and water reabsorption. Although GRK4 variants with elevated kinase activity have been associated with salt-sensitive or essential hypertension, this association has been inconsistent among different study populations. In addition, studies elucidating how GRK4 may modulate cellular signaling are sparse. In an analysis of how GRK4 affects the developing kidney, the authors found that GRK4 modulates mammalian target of rapamycin (mTOR) signaling. Loss of GRK4 in embryonic zebrafish causes kidney dysfunction and glomerular cysts. Moreover, GRK4 depletion in zebrafish and cellular mammalian models results in elongated cilia. Rescue experiments suggest that hypertension in carriers of GRK4 variants may not be explained solely by kinase hyperactivity; instead, elevated mTOR signaling may be the underlying cause.BackgroundG protein-coupled receptor kinase 4 (GRK4) is considered a central regulator of blood pressure through phosphorylation of renal dopaminergic receptors and subsequent modulation of sodium excretion. Several nonsynonymous genetic variants of GRK4 have been only partially linked to hypertension, although these variants demonstrate elevated kinase activity. However, some evidence suggests that function of GRK4 variants may involve more than regulation of dopaminergic receptors alone. Little is known about the effects of GRK4 on cellular signaling, and it is also unclear whether or how altered GRK4 function might affect kidney development.MethodsTo better understand the effect of GRK4 variants on the functionality of GRK4 and GRK4's actions in cellular signaling during kidney development, we studied zebrafish, human cells, and a murine kidney spheroid model.ResultsZebrafish depleted of Grk4 develop impaired glomerular filtration, generalized edema, glomerular cysts, pronephric dilatation, and expansion of kidney cilia. In human fibroblasts and in a kidney spheroid model, GRK4 knockdown produced elongated primary cilia. Reconstitution with human wild-type GRK4 partially rescues these phenotypes. We found that kinase activity is dispensable because kinase-dead GRK4 (altered GRK4 that cannot result in phosphorylation of the targeted protein) prevented cyst formation and restored normal ciliogenesis in all tested models. Hypertension-associated genetic variants of GRK4 fail to rescue any of the observed phenotypes, suggesting a receptor-independent mechanism. Instead, we discovered unrestrained mammalian target of rapamycin signaling as an underlying cause.ConclusionsThese findings identify GRK4 as novel regulator of cilia and of kidney development independent of GRK4's kinase function and provide evidence that the GRK4 variants believed to act as hyperactive kinases are dysfunctional for normal ciliogenesis.

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G-protein-coupled receptor kinase interacting protein-1 is required for pulmonary vascular development.

Publications

G-protein-coupled receptor kinase interacting protein-1 is required for pulmonary vascular development.

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