Int J Biochem Mol Biol 2013;4(1):41-53
Original Article
Functional silencing of guanylyl cyclase/natriuretic peptide receptor-A by microRNA
interference: analysis of receptor endocytosis
Naveen K Somanna, Amitabh C Pandey, Kiran K Arise, Vickie Nguyen, Kailash N Pandey
Department of Physiology, Tulane University Health Sciences Center School of Medicine, New Orleans, LA 70112, USA
Received December 31, 2012; Accepted January 15, 2013; Epub March 31, 2013; Published April 15, 2013
Abstract: Guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) is the principal receptor for the regulatory action of atrial and brain
natriuretic peptides (ANP and BNP) and an important effector molecule in controlling of extracellular fluid volume and blood pressure
homeostasis. We have utilized RNA interference to silence the expression of GC-A/NPRA gene (Npr1), providing a novel system to study the
internalization and trafficking of NPRA in intact cells. MicroRNA (miRNA)-mediated small interfering RNA (siRNA) elicited functional
gene-knockdown of NPRA in stably transfected human embryonic kidney 293 (HEK-293) cells expressing a high density of recombinant NPRA.
We artificially expressed three RNA polymerase II-driven miRNAs that specifically targeted the Npr1 gene, but shared no significant sequence
homology with any other known mouse genes. Reverse transcription-PCR (RT-PCR) and Northern blot analyses identified two highly efficient
Npr1 miRNA sequences to knockdown the expression of NPRA. The Npr1 miRNA in chains or clusters decreased NPRA expression more
than 90% as compared with control cells. ANP-dependent stimulation of intracellular accumulation of cGMP and guanylyl cyclase activity of
NPRA were significantly reduced in Npr1 miRNA-expressing cells by 90-95% as compared with control cells. Treatment with Npr1 miRNA
caused a drastic reduction in the receptor density subsequently a deceased internalization of radiolabeled 125I-ANP-NPRA ligand-receptor
complexes. Only 12%-15% of receptor population was localized in the intracellular compartments of microRNA silenced cells as compared to
70%-80% in control cells. (IJBMB1212005).
Keywords: Atrial natriuretic peptide, guanylyl cyclase/natriuretic peptide receptor-A, gene-knockdown, receptor internalization, human
embry-onic kidney cells, microRNA
Address correspondence to: Dr. Kailash N Pandey, Department of Physiology, SL-39, Tulane University Health Sciences Center, 1430 Tulane
Ave, New Orleans, LA 70112, USA. Phone: 504-584-1628; Fax: 504-584-2675; E-mail: kpandey@tulane.edu
Original Article
Functional silencing of guanylyl cyclase/natriuretic peptide receptor-A by microRNA
interference: analysis of receptor endocytosis
Naveen K Somanna, Amitabh C Pandey, Kiran K Arise, Vickie Nguyen, Kailash N Pandey
Department of Physiology, Tulane University Health Sciences Center School of Medicine, New Orleans, LA 70112, USA
Received December 31, 2012; Accepted January 15, 2013; Epub March 31, 2013; Published April 15, 2013
Abstract: Guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) is the principal receptor for the regulatory action of atrial and brain
natriuretic peptides (ANP and BNP) and an important effector molecule in controlling of extracellular fluid volume and blood pressure
homeostasis. We have utilized RNA interference to silence the expression of GC-A/NPRA gene (Npr1), providing a novel system to study the
internalization and trafficking of NPRA in intact cells. MicroRNA (miRNA)-mediated small interfering RNA (siRNA) elicited functional
gene-knockdown of NPRA in stably transfected human embryonic kidney 293 (HEK-293) cells expressing a high density of recombinant NPRA.
We artificially expressed three RNA polymerase II-driven miRNAs that specifically targeted the Npr1 gene, but shared no significant sequence
homology with any other known mouse genes. Reverse transcription-PCR (RT-PCR) and Northern blot analyses identified two highly efficient
Npr1 miRNA sequences to knockdown the expression of NPRA. The Npr1 miRNA in chains or clusters decreased NPRA expression more
than 90% as compared with control cells. ANP-dependent stimulation of intracellular accumulation of cGMP and guanylyl cyclase activity of
NPRA were significantly reduced in Npr1 miRNA-expressing cells by 90-95% as compared with control cells. Treatment with Npr1 miRNA
caused a drastic reduction in the receptor density subsequently a deceased internalization of radiolabeled 125I-ANP-NPRA ligand-receptor
complexes. Only 12%-15% of receptor population was localized in the intracellular compartments of microRNA silenced cells as compared to
70%-80% in control cells. (IJBMB1212005).
Keywords: Atrial natriuretic peptide, guanylyl cyclase/natriuretic peptide receptor-A, gene-knockdown, receptor internalization, human
embry-onic kidney cells, microRNA
Address correspondence to: Dr. Kailash N Pandey, Department of Physiology, SL-39, Tulane University Health Sciences Center, 1430 Tulane
Ave, New Orleans, LA 70112, USA. Phone: 504-584-1628; Fax: 504-584-2675; E-mail: kpandey@tulane.edu
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