This study reports, we believe for the first time, the existence of a functional receptor of renin. The evidence that N14F cDNA is identical to the receptor of renin is as follows: (a) The N14F protein expressed in vitro is able to bind renin in coprecipitation experiments; (b) cells transfected with N14F cDNA construct and expressing the protein bind renin with high affinity, and the binding is specific for renin and prorenin; (c) renin binding induces the activation of MAP kinases ERK1 and ERK2 associated with a phosphorylation of tyrosine and serine residues.
The localization of the receptor in the mesangium of glomeruli suggests that this receptor is the one we described on human mesangial cells in culture. In primary mesangial cells (21
) we have observed a molecular weight of 70–80 kDa for the receptor, whereas the cloned renin receptor here is 45 kDa. This discrepancy may be explained by a dimerization of the receptor induced by renin binding as described for receptors of the tyrosine-kinase family, which are also single-transmembrane domain receptors (32
), or a complex formation with another, yet-unidentified protein. Part of this clone was identical to the previously reported “M8-9,” a truncated protein of 8.9 kDa that copurified with a proton-ATPase of chromaffin granule membranes (33
). While the sequencing of our N14F clone was completed, two identical cDNA sequences from human fetal brain and human hypothalamus appeared in Genbank (DKFZ p56400582, accession number AL049929.1 and AF248966), confirming the high level of expression of the receptor in the human CNS.
The physiological plasma concentration of renin is in the picomolar range, but it is admitted that in tissues, especially in interstitial fluids, the renin concentration may be 100-fold greater. Since we found that the dissociation constant of the renin-receptor complex was in the nanomolar range, we postulated that it could not be considered as a binding protein responsible for retaining renin in the interstitium because only about 1% of the interstitial renin (10 pM) would be bound by the receptor. Alternatively, it could function as an effective receptor. Therefore, we looked for [Ca2+] change and for cyclic AMP and MAP kinases ERK1(p44)/ERK2(p42) activation. Our results showed that renin binding did not modify either intracellular calcium or cAMP, but provoked a rapid activation of the ERK1/ERK2 pathway, associated with a phosphorylation of serine and tyrosine residues. The presence of either an ACE inhibitor or an AT1 receptor antagonist during the incubation of transformed cells with renin confirms that these events are independent of a possible generation or action of Ang II. The pathway involved in the tyrosine phosphorylation of the receptor, the respective roles of serine and tyrosine phosphorylation in the receptor function, and the early phase of renin signal are actually under investigation.
We have shown that receptor-bound renin activates angiotensinogen with kinetics different from those observed for renin in solution and, in particular, we showed a reduction in the Km
for angiotensinogen from 1 μM in solution to 0.15 μM with receptor-bound renin. This Km
is significantly below the normal plasma concentration of angiotensinogen, approximately 1 μM (29
), suggesting that conversion of angiotensinogen by receptor-bound renin may be of physiological importance, especially in tissues in which the concentration of angiotensinogen is much lower than in plasma. We hypothesize that receptor-bound renin is able to activate angiotensinogen while it is actually bound to the cell surface and with higher efficiency than renin in solution. The slight decrease of kcat
(1.5-fold reduction) observed concomitantly might reflect the constraints imposed by the immobilization of renin on the receptor. The overall catalytic efficiency kcat
increased four to five times, indicating that the cell surface is an important site for angiotensinogen activation. Taken together with the recent report of the existence of a vascular smooth muscle chymase (34
), our results suggest that the smooth muscle cell surface may play an essential role in tissue generation of Ang II. The binding of renin to its receptor, thereby increasing angiotensinogen cleavage efficiency, would facilitate Ang I cleavage in Ang II by vascular smooth muscle chymase.
We also hypothesized that bound prorenin would activate angiotensinogen. Although our prorenin preparation was reactive against Ab’s to the active site of renin, suggesting that some molecules have an accessible active site, prorenin showed very little Ang I–forming activity when incubated at pH 7.4 for 4 hours. However, the extent of Ang I generated by prorenin bound to membranes was comparable to the extent of Ang I generated by fully active renin under these experimental conditions. Whether the increase of the catalytic activity of membrane-bound prorenin should be attributed to the fraction of prorenin with an already exposed active site or to inactive prorenin that underwent conformational changes induced by receptor binding is an important issue to investigate in the near future. Supporting evidence for catalytically active prorenin in vivo,
in the absence of prorenin cleavage, exists in the literature (35
). Methot et al. generated double-transgenic mice expressing human angiotensinogen and a mutated, noncleavable human prorenin in the pituitary gland (35
). These animals have elevated pituitary Ang I content in the absence of prorenin cleavage as shown by Western blot analysis, indicating that prorenin was enzymatically active even with the prosegment still in place.
Knockout mice for angiotensinogen, Ang converting enzyme, Ang II receptors, and nullizygotes for the renin gene, display low blood pressure and severe renal vascular lesions associated with high or low plasma renin concentrations (36
). The vascular lesions have been attributed to the absence of Ang II during development, but these data do not exclude a role of renin in the pathogenesis of vascular lesions. Moreover, rats expressing the prorenin transgene exclusively in the liver suffer severe nephroangiosclerosis, cardiac and aortic hypertrophy, and liver fibrosis in the absence of hypertension (40
). This observation suggests that the effects observed in vitro on the activation of ERK1/ERK2 pathway involved in cell hypertrophy and proliferation, as well as the cellular hypertrophy and the increase of PAI1 (21
) synthesis, may be relevant in vivo.
In conclusion, our results show that renin receptor exerts dual effects. The renin receptor is able to trigger intracellular signal by activating the ERK1/ERK2 pathway, and it also acts as a cofactor by increasing the efficiency of angiotensinogen cleavage by receptor-bound renin, therefore facilitating Ang II generation and action on a cell surface. This novel concept may provide a new approach to a better understanding of the pathogenesis of vascular diseases associated with RAS activation.