Characterization of AT4 receptor from bovine aortic endothelium with photosensitive analogues of angiotensin IV.

Newly developed photosensitive analogues of AngIV were used to characterize the AT4 receptor of bovine aortic endothelial cells. The photoactivatable AngIV analogues [N3-Phe6]AngIV and [Bpa6]AngIV displayed high affinities for AT4 receptor, with IC50's of 3.7 +/- 0.3 and 19.1 +/- 3.5 nM, respectively. The radioiodinated ligands showed a good efficiency of photoaffinity labeling demonstrated by high proportions (60-75%) of acid-resistant binding. Covalently labeled receptor was solubilized under reducing or nonreducing conditions and subjected to SDS-PAGE. Under nonreducing conditions, autoradiographies revealed a major band of Mr 186 +/- 2 kDa and a minor band of Mr 241 +/- 6 kDa. The labeling of these bands was completely abolished in the presence of 10 microM AngIV. Under reducing conditions, only the low Mr 186 kDa band was revealed. After endoglycosidase digestion with an enzyme that cleaves N-linked saccharides, the Mr of the denatured AT4 receptor was decreased by 31% to a value of 129 +/- 10 kDa. Kinetic studies revealed a stepwise process of AT4 receptor deglycosylation by endoglycosidase F, suggesting at least two different sites of N-linked saccharides. Mild trypsin treatment of photolabeled endothelial cell membranes released a large fragment of Mr 177 +/- 3 kDa which accounts for about 95% of the whole receptor molecular mass. These results demonstrate that [N3-Phe6]AngIV and [Bpa6]AngIV are very efficient tools for selective photoaffinity labeling of AT4 receptor. We have shown that AT4 receptor is a 186 kDa integral membrane glycoprotein with a very large extracellular domain. These properties are consistent with those of a growth factor or cytokine receptor.

Study on the functionality and molecular properties of the AT4 receptor.

Whereas the role of angiotensin II (Ang II) has been clarified in numerous tissues and cell types, the physiological relevance of its C-terminal (3-8) degradation fragment, angiotensin IV (Ang IV), is unclear. Previously, we characterized a specific binding site for Ang IV in the bovine adrenal cortex and on bovine aortic endothelial cells (BAEC). In the present study, we tried to assess the functionality and mechanism of action of this receptor for Ang IV (AT4 receptor). Our results revealed that none of the classical second messengers (i.e., cAMP, Ca2+, inositol phosphates, nitric oxide or arachidonic acid derivatives) was modified significantly during acute (less than 1 h) stimulation of cells with Ang IV. Under normal culture conditions, BAEC efficiently internalized 125I-Ang IV. After a 2 h incubation at 37 degrees C, acid-resistant binding corresponded to about 50% of total cell-associated radioactivity. This rapid internalization process suggests that the AT4 receptor is a functional protein. With a photoaffinity labeling approach, we revealed some properties of the AT4 receptor that are consistent with those of a growth factor or cytokine receptor.