Eric Clauser

Angiotensin II type 1 receptor gene polymorphisms in human essential hypertension.

We conducted the present study to determine whether the angiotensin II type I receptor (AT1) gene might be implicated in human essential hypertension by using case-control and linkage studies. The entire coding and 3 untranslated regions of the AT1 receptor gene (2.2 kb) were amplified by polymerase chain reaction and submitted to single-strand conformation polymorphism in 60 hypertensive subjects with a familial susceptibility. We identified five polymorphisms (T573-->C, A1062-->G, A1166-->C, G1517-->T, and A1878-->G). However, no mutations that alter the encoded amino acid sequence were detected. A case-control study performed on white hypertensive (n = 206; blood pressure, 168 +/- 16/103 +/- 9 mm Hg) and normotensive (n = 298; blood pressure, 122 +/- 10/75 +/- 9 mm Hg) subjects using three of five polymorphisms showed a significant increase in allelic frequency of C1166 in hypertensive subjects (0.36 versus 0.28 for normotensive subjects, chi 2 = 6.8, P < .01). Frequencies for the alleles of the other two polymorphisms (T573-->C, A1878-->G) were similar in both groups. We performed a linkage study using the affected sib pair method and a highly polymorphic marker of the AT1 receptor gene. There was no evidence for linkage in 267 sib pairs analyzed from 138 pedigrees. These findings would be compatible with a common variant of the AT1 receptor imparting a small effect on blood pressure; further studies will be needed to address this possibility.

Polar Residues in the Transmembrane Domains of the Type 1 Angiotensin II Receptor Are Required for Binding and Coupling RECONSTITUTION OF THE BINDING SITE BY CO-EXPRESSION OF TWO DEFICIENT MUTANTS

Type 1 angiotensin receptors (AT) are G-protein coupled receptors, mediating the physiological actions of the vasoactive peptide angiotensin II. In this study, the roles of 7 amino acids of the rat AT receptor in ligand binding and signaling were investigated by performing functional assays of individual receptor mutants expressed in COS and Chinese hamster ovary cells. Substitutions of polar residues in the third transmembrane domain with Ala indicate that Ser, Ser, and Ser are not essential for maintenance of the angiotensin II binding site. Replacement of Asn or Ser does not alter the binding affinity for peptidic analogs, but modifies the ability of the receptor to interact with AT (DuP753)- or AT (CGP42112A)-specific ligands. These 2 residues are probably involved in determining the binding specificity for these analogs. The absence of G-protein coupling to the Ser mutant suggests that this residue, in addition to previously identified residues, Asp and Tyr, participates in the receptor activation mechanism. Finally, Lys (third helix) and Lys (fifth helix) mutants do not bind angiotensin II or different analogs. Co-expression of these two deficient receptors permitted the restoration of a normal binding site. This effect was not due to homologous recombination of the cDNAs but to protein trans-complementation.

CLONING AND CHARACTERIZATION OF THE HUMAN V3 PITUITARY VASOPRESSIN RECEPTOR

Arginine‐vasopressin (AVP) plays a determinant role in the normal ACTH response to stress in mammals. We cloned a human cDNA coding a 424 amino acid G‐protein coupled receptor structurally related to the vasopressin/oxytocin receptor family. When expressed in COS cells, this receptor binds AVP with a high affinity (K d = 0.55 ± 0.13 nM) and is functionally coupled to phospholipase C. Competition studies with peptidic or non peptidic AVP analogues reveal that it is pharmacologically distinct from V1a and V2 AVP receptors and therefore it is designated V3. RT‐PCR analysis shows that the human V3 receptor is expressed in normal pituitary and also in kidney, but is undetectable in liver, myometrium and adrenal gland. Northern blot analysis reveals a ∼4.8 kb messenger in human corticotropic pituitary adenomas.

Identification of the Rat Adapter Grb14 as an Inhibitor of Insulin Actions

We cloned by interaction with the β-subunit of the insulin receptor the rat variant of the human adapter Grb14 (rGrb14). rGrb14 is specifically expressed in rat insulin-sensitive tissues and in the brain. The binding of rGrb14 to insulin receptors is insulin-dependent in vivo in Chinese hamster ovary (CHO) cells overexpressing both proteins and importantly, in rat liver expressing physiological levels of proteins. However, rGrb14 is not a substrate of the tyrosine kinase of the receptor. In the two-hybrid system, two domains of rGrb14 can mediate the interaction with insulin receptors: the Src homology 2 (SH2) domain and a region between the PH and SH2 domains that we named PIR (forphosphorylated insulin receptor-interactingregion). In vitro interaction assays using deletion mutants of rGrb14 show that the PIR, but not the SH2 domain, is able to coprecipitate insulin receptors, suggesting that the PIR is the major binding domain of rGrb14. The interaction between rGrb14 and the insulin receptors is almost abolished by mutating tyrosine residue Tyr1150 or Tyr1151 of the receptor. The overexpression of rGrb14 in CHO-IR cells decreases insulin stimulation of both DNA and glycogen synthesis. These effects are accompanied by a decrease in insulin-stimulated tyrosine phosphorylation of IRS-1, but insulin receptor autophosphorylation is unaltered. These findings suggest that rGrb14 could be a new downstream signaling component of the insulin-mediated pathways.

Immunocytochemical localization of angiotensinogen in rat liver and kidney

SummaryThe renin substrate, angiotensinogen, was localized by immunocytochemistry in liver and kidney of normal rats by the use of an antiserum directed against pure rat angiotensinogen. This substrate was also examined in rats after bilateral nephrectomy, which is known to increase plasma angiotensinogen, and in rats treated with colchicine, which inhibits serum protein secretion.In normal rat liver, light microscopy showed the presence of immunoreactive material in a very few cells. The number of stained hepatocytes rose in rats treated with colchicine or after bilateral nephrectomy. Immuno-staining increased further when rats were both nephrectomized and colchicine treated.In the kidney, angiotensinogen was specifically located as granular formations in nephrocytes of the proximal tubule but never in the granular cells of the juxtaglomerular apparatus. The localization of these granular formations under the brush border suggests that angiotensinogen is reabsorbed from the glomerular ultrafiltrate rather than synthesized in the kidney.

Role of angiotensinogen in blood pressure homeostasis.

The role of angiotensinogen ID blood pressure control was assessed in nonnotensive rate by observing the changes resulting from inhibition by specific rat angiotensinogen antiserum. The antiserura decreased blood pressure in rats on normal sodium as well as sodium-free diets (respectively ΔBP = −30 ± 6 mm Hg and −42 ± 8 mm Hg). In binephrectomlzed sodium-replete rats, administration of antiserum did not reduce blood pressure, whereas in sodium-depleted animals it slightly decreased blood pressure by 11 ± 3 mm Hg. These results suggest that angiotensinogen participates in the regulation of blood pressure in normotensive rats, even in the sodium-replete state. (Hypertension 4: 185–189, 1982)

The C-terminal third intracellular loop of the rat AT1A angiotensin receptor plays a key role in G protein coupling specificity and transduction of the mitogenic signal.

To identify the role(s) of the third intracellular loop of the angiotensin II (AngII) type 1A (AT1A) receptor in G protein coupling specificity and receptor activation, several chimerae were constructed and characterized. The cDNA sequence encoding the C-terminal segment of the third intracellular loop of the AT1A receptor (residues 234–240) was replaced with the homologous regions of the α1B adrenergic (α1B-AR), the β2 adrenergic (β2-AR), and the AngII type 2 (AT2) receptors. These chimeric receptors were stably expressed in Chinese hamster ovary cells, and their pharmacological and functional properties were characterized, including AngII-induced inositol phosphate and cyclic AMP (cAMP) productions, [3H]thymidine incorporation into DNA, and internalization. The affinities of these chimeric receptors for [Sar1]AngII, [Sar1,Ile8]AngII, and losartan were essentially normal; however, the affinity of these mutants was increased by a factor of 10–40 for the AT2-specific ligand CGP42112A. The functional properties of the α1B-AR chimera were essentially identical to those of the wild type AT1A receptor. On the other hand, replacement with the β2-AR segment produced a partial reduction of the inositol phosphate production, a measurable AngII-induced cAMP accumulation, a reduced internalization, and a total impairment to transduce the mitogenic effect of AngII. The AT2 chimera presented a normal internalization, but was inactive in all the other functional tests. In conclusion, the distal segment of the third intracellular loop of the rat AT1A receptor plays a pivotal role in coupling selectivity and receptor signaling via G protein(s) as well as in the activation of the specific signaling pathways involved in the mitogenic actions of AngII.

A recombinant rat vascular AT1 receptor confers growth properties to angiotensin II in Chinese Hamster Ovary cells

A rat vascular AT1 receptor cDNA has been stably expressed into Chinese Hamster Ovary cells and the resulting recombinant AT1a receptor has been functionally characterized. This receptor binds 125I Sar1-angiotensin II with an affinity of 0.9 nM and the displacement of this ligand by a series of peptidic and nonpeptidic analogs is shown. Binding of angiotensin II to this receptor causes a rapid increase in inositol phosphate production, whereas this effect is not observed in nontransfected cells. Des-aspartyl1 angiotensin II and at a lesser extent angiotensin I are also able to produce an increase in inositol phosphates. More importantly, the actions of angiotensin II on cell division were clearly demonstrated in this model, since angiotensin II is able to stimulate DNA synthesis by 400% and double the cell population of the transfected cells in 36 hours in the absence of any other growth factor, whereas no effect is observed in nontransfected cells.

V3 Vasopressin Receptor and Corticotropic Phenotype in Pituitary and Nonpituitary Tumors

Pituitary corticotropic cells express a specific vasopressin receptor, called V1b or V3, through which vasopressin stimulates corticotropin secretion. We recently cloned a cDNA coding for this receptor and showed that it belongs to the G protein-coupled receptor family. V3 mRNA is readily detected by RT-PCR in normal human pituitaries and corticotropic pituitary adenomas but not in PRL or GH-secreting adenomas, thus demonstrating that, like POMC itself and the CRH receptor, V3 is a marker of the corticotropic phenotype. Nuclease protection experiments suggest that V3 is overexpressed in some corticotropic adenomas, and thus may play a role in tumor development by activating the phospholipase C-signalling pathway. In addition analysis of its expression in nonpituitary neuroendocrine tumors showed a striking association with carcinoids of the lung responsible for the ectopic ACTH syndrome.

Vasopressin receptors modulate the pharmacological phenotypes of cushing's syndrome

We have examined the expression profiles of the different vasopressin receptors (V1, V2, V3) that can be expressed in the three different types of tumors associated with Cushings syndrome. V3 (V1b) receptor cDNA was cloned from a pituitary tumor responsible for Cushings disease. We show that it is overexpressed in these tumors and can respond to DD-AVP. High expression of the V3 receptor on highly differentiated, ACTH-secreting, bronchial carcinoid tumors explain why these non-pituitary tumors occasionally respond to vasopressin, mimicking a pituitary-like behavior. A retrospective analysis showed that vasopressin induced an ACTH-independent cortisol rise in 27% of the adrenocortical tumors responsible for Cushings syndrome. V1 mRNA was detected in normal adrenal cortex and in all tumors. Adenomas had significantly higher levels than carcinomas. V1 mRNA levels were higher in responders than in non-responders. One adenoma which had a brisk cortisol response in vivo, also had in vitro cortisol responses that were inhibited by a specific V1 antagonist. In situ hybridization showed the presence of V1 mRNA in the normal human adrenal cortex where the signal predominated in the compact cells of the zona reticularis. A positive signal was also present in the tumors with high V1 mRNA levels determined by RT-PCR; its distribution pattern was heterogeneous and showed preferential association with compact cells. High-and not ectopic-expression of the V1 receptor occurs in a minority of adrenal cortical tumors which become directly responsive to vasopressin stimulation.