Transduction of a functional domain of the AT1 receptor in neurons by HIV-Tat PTD.
Despite advances in transgenic and gene transfer technologies, in vivo structure-function studies of the angiotensin II type I receptor (AT1R) have revealed limited information on the diverse actions of angiotensin II. Our objective in the present study was to determine if protein transduction technology with the use of the HIV-Tat protein transduction domain could fill this gap. Recombinant HIV-Tat protein transduction domain fused to EGFP and to the third intracellular loop of the AT1R was expressed. Incubation of hypothalamus and brainstem neurons with this peptide indicated an efficient transport of the protein to most of the cells. This transduction was accompanied by an increase in neuronal firing rate, an effect similar to that observed with angiotensin II stimulation of the neuronal AT1R. The characteristics of the chronotropic effects of recombinant third intracellular loop and its synthetic counterpart were similar and comparable to the effects of angiotensin II on these neurons. In addition, in the presence of the protein kinase C inhibitor calphostin C, the peptide failed to increase firing rate. These observations demonstrated that transduction of neurons with the third intracellular loop of the AT1R produces chronotropic effects similar to those induced by angiotensin II. The data suggests that protein transduction technology could be useful for in vivo AT1R domain transduction.
Vázquez
Jorge
J
Department of Physiology and Functional Genomics and the University of Florida, McKnight Brain Institute, Gainesville 32610, USA.
Sun
Chengwen
C
Du
Jianqing
J
Fuentes
Lucía
L
Sumners
Colin
C
Raizada
Mohan K
MK
D000200:Action Potentials; D000818:Animals; D018920:Coculture Techniques; D015696:Gene Products, tat / Q000737:chemistry* / Q000235:genetics; D049452:Green Fluorescent Proteins; D006678:HIV / Q000737:chemistry; D008164:Luminescent Proteins / Q000235:genetics; D009474:Neurons / Q000378:metabolism* / Q000502:physiology; D015202:Protein Engineering / Q000379:methods*; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051381:Rats; D011921:Rats, Inbred WKY; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D008164:Luminescent Proteins; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins; D011493:Protein Kinase C