Importance of the hydrophobic residues of loop extracellular III of the AT1 receptor of angiotensin II in the interaction with its ligands

The role of the external third loop (EC-3) of the angiotensin II (AII) AT1, receptor for the interaction with its ligand was investigated by performing triple simuitaneous mutation of L262, L265 and L268 for Asp ([L3D3]AT1), in addition to two single mutantions of Leu265 [L265A]AT1, and L268, [L268R]AT1. The mutants were permanently transfected to Chinese hamster ovary cells (CHO). Binding assays showed that [L3D3]AT1, had very low affinity to Ali and DuP753 whereas its binding to [Sar1,Leu8-AII was not detectable. However no great changes were observed towards [L265A]AT1 and to [L268R]AT1 which produced lC5O values similar to that obtained with wild type receptor.(control) The inositol phosphate responses to AII was much drastically reduced in [L3D3]AT1. Therefore the ED50 value and the maximal effect were significantiy lower that those of wild type. It was also observed that the expression of [L3D3]AT1 was drasticaily reduced. However [L268R]AT1 and [L265A]AT1 were able to stimulate inositol phosphate formation and its maximal effect not significantly different from the controls. These effects could be only detected when multiple but not single mutations were performed. Moreover the hydrophobic residues located in this N-terminal region of EC-3 should be substituted for residues bearing charges. From these results and from the literature it is suggested that AII was less affected than [Sar1,Leu8]-AII due to its strong interactions with transmembrane residues of the receptor while the extracellular residues seem to be more important to the interactions of [Sar1,Leu8]-AII with the receptor. This could explain why [Sar1,Leu8]-AII was more affected by the mutations of [L3D3]AT1 than AII. On the other hand as DuP753 partially share some residues such as Lysl99 and His256 for interaction with AII this could explain the similar effect exerted by [L3D3]AT1 on both ligands. These resuits suggest that the hydrophobic residues located in the EC-3, close to heiix VI are critical to maintain the stability of ligand bindingBV UNIFESP: Teses e dissertaçõe

Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags

Transcribed sequences in the human genome can be identified with confidence only by alignment with sequences derived from cDNAs synthesized from naturally occurring mRNAs. We constructed a set of 250,000 cDNAs that represent partial expressed gene sequences and that are biased toward the central coding regions of the resulting transcripts. They are termed ORF expressed sequence tags (ORESTES). The 250,000 ORESTES were assembled into 81,429 contigs. Of these, 1,181 (1.45%) were found to match sequences in chromosome 22 with at least one ORESTES contig for 162 (65.6%) of the 247 known genes, for 67 (44.6%) of the 150 related genes, and for 45 of the 148 (30.4%) EST-predicted genes on this chromosome. Using a set of stringent criteria to validate our sequences, we identified a further 219 previously unannotated transcribed sequences on chromosome 22. Of these, 171 were in fact also defined by EST or full length cDNA sequences available in GenBank but not utilized in the initial annotation of the first human chromosome sequence. Thus despite representing less than 15% of all expressed human sequences in the public databases at the time of the present analysis, ORESTES sequences defined 48 transcribed sequences on chromosome 22 not defined by other sequences. All of the transcribed sequences defined by ORESTES coincided with DNA regions predicted as encoding exons by genscan. (http://genes.mit.edu/GENSCAN.html)