Antidiuresis, the recovery of water from the lumen of the renal collecting tubule, is regulated by the hypothalamic release of antidiuretic hormone (ADH), which binds to specific receptors on renal collecting tubule cells, stimulates adenylyl cyclase and promotes the cyclic AMP-mediated incorporation of water pores into the luminal surface of these cells. We report here the isolation of the human ADH receptor gene using a genomic expression cloning approach. The gene was used to clone the complementary DNA from a human renal library. The deduced amino-acid sequence of the receptor yields a hydropathy profile characteristic of receptors with seven putative transmembrane regions. This and the comparison with other cloned receptors indicates that the ADH receptor is a member of the superfamily of G-protein-coupled receptors.

Previous studies indicated that partial agonists cause less desensitization of the ␤ 2 -adrenergic receptor (␤AR) than full agonists; however, the molecular basis for this in intact cells has not been investigated. In the present work, we have determined the rates of desensitization, internalization, and phosphorylation caused by a series of ␤AR agonists displaying a 95-fold range of coupling efficiencies. These studies were performed with HEK-293 cells overexpressing the ␤AR with hemagglutinin and 6-histidine epitopes introduced into the N and C termini, respectively. This modified ␤AR behaved identically to the wild type receptor with regard to agonist K d , coupling efficiency, and desensitization. The coupling efficiencies for ␤AR agonist activation of adenylyl cyclase relative to epinephrine (100%) were 42% for fenoterol, 4.9% for albuterol, 2.5% for dobutamine, and 1.1% for ephedrine. At concentrations of these agonists yielding >90% receptor occupancy, the rate and extent (0 -30 min) of agonist-induced desensitization of ␤AR activation of adenylyl cyclase followed the same order as coupling efficiency, i.e. epinephrine > fenoterol > albuterol > dobutamine > ephedrine. The rate of internalization of the ␤AR with respect to these agonists also followed the same order as the desensitization and exhibited a slight lag. Like internalization and desensitization, ␤AR phosphorylation exhibited a dependence on agonist strength. The two strongest agonists, epinephrine and fenoterol, provoked 11-13-fold increases in the level of ␤AR phosphorylation after just 1 min, whereas the weak agonists dobutamine and ephedrine caused only 3-4-fold increases, similar to levels induced by cAMP-dependent protein kinase activation with forskolin. With longer treatment times, the level of ␤AR phosphorylation declined with strong agonists, but it progressively increased with the weaker partial agonists, such that after 30 min the -fold elevation with epinephrine (6.2 ؎ 0.82) was not appreciably different from ephedrine (5.0 ؎ 0.96) and significantly less than that caused by albuterol (10.4 ؎ 1.7). In summary, our results demonstrate an excellent proportionality between the agonist strength and agonist-induced desensitization, internalization, and the rapid initial phase of phosphorylation. The data support the hypothesis that increasing agonist-coupling efficiency primarily affects desensitization by increasing the rate of ␤ARK phosphorylation of the ␤AR.The adenylyl cyclase-coupled ␤AR 1 system has served as a model system for the study of the phenomenon of desensitization of G-protein-coupled receptors (1-5). Desensitization is functionally defined as an attenuation of hormonal responsiveness upon agonist stimulation. There are four currently known mechanisms of agonist-induced desensitization that appear to have physiological significance: receptor sequestration/internalization (1), ␤AR kinase (␤ARK) phosphorylation of serines and threonines on the ␤AR C terminus (3, 5), cAMP-dependent protein kinase (PKA) phosphorylation of ␤AR ...

The human beta(2)-adrenergic receptor (betaAR) is rapidly desensitized in response to saturating concentrations of agonist by G protein-coupled receptor kinases (GRKs) and cAMP-dependent protein kinase A (PKA) phosphorylation of the betaAR, followed by beta-arrestin binding and receptor internalization. betaAR sites phosphorylated by GRK in vivo have not yet been identified. In this study, we examined the role of the carboxyl terminal serines, 355, 356, and 364, in the GRK-mediated desensitization of the betaAR. Substitution mutants of these serine residues were constructed in which either all three (S355,356,364A), two (S355,356A and S356, 364A), or one of the serines (S356A and S364A) were modified. These mutants were constructed in a betaAR in which the serines of the PKA consensus site were substituted with alanines (designated PKA(-)) to eliminate any PKA contribution to desensitization, and they were stably transfected into human embryonic kidney 293 cells. Treatment of the PKA(-) mutant with 10 microM epinephrine for 5 min caused a 3. 5-fold increase in the EC(50) value and a 42% decrease in the V(max) value for epinephrine stimulation of adenylyl cyclase. Substitution of all three serines completely inhibited the epinephrine-induced shift in the EC(50). Both double mutants, S355,356A and S356,364A, showed a nearly complete loss of the EC(50) shift, whereas the single substitutions, S356A and S364A, caused only a slight decrease in desensitization. None of the mutations altered the epinephrine-induced decrease in V(max,) which seems to be downstream of the receptor. The triple mutation caused a 45% decrease in epinephrine-induced internalization and a 90 to 95% reduction in phosphorylation of the betaAR relative to the PKA(-) (1.9+/- 0.2- and 16.6+/-3.8-fold phosphorylation over basal, respectively). The double mutants caused an intermediate reduction in internalization (20-21%) and phosphorylation (43-52%). None of the serine mutations altered the rate of betaAR recycling. Our data demonstrate that the cluster of serines within the 355 to 364 betaAR domain confer the rapid, GRK-mediated, receptor-level desensitization of the betaAR.

Antidiuretic hormone (arginine vasopressin) binds to and activates V2 receptors in renal collecting tubule cells. Subsequent stimulation of the Gs/adenylyl cyclase system promotes insertion of water pores into the luminal membrane and thereby reabsorption of fluid. In congenital nephrogenic diabetes insipidus (CNDI), an X-linked recessive disorder, the kidney fails to respond to arginine vasopressin. Here we report that an affected male of a family with CNDI has a deletion in the open reading frame of the V2 receptor gene, causing a frame shift and premature termination of translation in the third intracellular loop of the receptor protein. A normal receptor gene was found in the patient's brother. Both the normal and the mutant allele were detected in his mother. A different mutation, causing a codon change in the third transmembrane domain of the V2 receptor, was found in the open reading frame of an affected male but not in the unaffected brother belonging to another family suffering from CNDI.

1 Partial agonists of the b 2 -adrenoceptor which activate adenylyl cyclase are widely used as bronchodilators for the relief of bronchoconstriction accompanying many disease conditions, including bronchial asthma. The bronchodilator salmeterol has both a prolonged duration of action in bronchial tissue and the ability to reassert this activity following the temporary blockade of human b 2 -adrenoceptors with antagonist. 2 We have compared the activation and desensitization of human b 2 -adrenoceptor stimulation of adenylyl cyclase induced by salmeterol, adrenaline and salbutamol in a human lung epithelial line, BEAS-2B, expressing b 2 -adrenoceptor levels of 40 ± 70 fmol mg 71 , and in human embryonic kidney (HEK) 293 cell lines expressing 2 ± 10 pmol mg 71 . The e cacy observed for the stimulation of adenylyl cyclase by salmeterol was only %10% of that observed for adrenaline in BEAS-2B cells expressing low levels of b 2 -adrenoceptor, but similar to adrenaline in HEK 293 cells expressing very high levels of receptors. Salmeterol pretreatment of these cells induced a rapid and stable activation of adenylyl cyclase activity which resisted extensive washing and b 2 -adrenoceptor antagonist blockade, consistent with binding to a receptor exosite and/or to partitioning into membrane lipid. 3 The desensitization and internalization of b 2 -adrenoceptors induced by the partial agonists salmeterol and salbutamol were considerably reduced relative to the action of adrenaline. Consistent with these observations, the initial rate of phosphorylation of the receptor induced by salmeterol and salbutamol was much reduced in comparison to adrenaline. 4 Our data suggest that the reduction in the rapid phase of desensitization of b 2 -adrenoceptors after treatment with salmeterol or salbutamol is caused by a decrease in the rate of b 2 -adrenoceptor kinase (bARK) phosphorylation and internalization. In contrast, the rate of cyclic AMP-dependent protein kinase (PKA)-mediated phosphorylation by these partial agonists appears to be similar to adrenaline.