Aspartate138 is required for the high-affinity ligand binding site but not for the low-affinity binding site of the beta1-adrenoceptor.

The beta(1)-adrenoceptor "two-site ligand binding hypothesis" was investigated by comparing the pharmacological activities of the receptor with an Asp to Glu mutation of amino acid 138 after transient transfection into CHO cells. The high-affinity binding of (-)-[(3)H]-CGP12177 (p K(D)=9.4) and binding inhibition by (-)-isoprenaline (p K(i)=6.2), observed with Asp138-beta(1)-adrenoceptors, were absent at Glu138-beta(1)-adrenoceptors. (-)-[(3)H]-CGP12177 bound with a p K(D)=7.6 to Glu138-beta(1)-adrenoceptors and (-)-isoprenaline enhanced binding, probably allosterically. Glu138-beta(1)-adrenoceptors compared with Asp138-beta(1)-adrenoceptors showed a 500,000-fold decrease in cyclic AMP-enhancing potency by (-)-isoprenaline and antagonism by (-)-bupranolol (1 microM) was abolished. At Glu138-beta(1)-adrenoceptors, the agonist potency of (-)-CGP12177, compared with (-)-isoprenaline was reduced five-fold, but the antagonism by (-)-bupranolol (p K(B)=7.1) was not significantly changed, compared with Asp138-beta(1)-adrenoceptor. Thus, Asp138 of the beta(1)-adrenoceptor is essential for the binding of (-)-isoprenaline, (-)-bupranolol and (-)-CGP12177 to a high-affinity site, but not for the binding of (-)-CGP12177 and (-)-bupranolol to a low-affinity site.

Binding of (-)-[3H]-CGP12177 at two sites in recombinant human beta 1-adrenoceptors and interaction with beta-blockers.

To verify the hypothesis that the non-conventional partial agonist (-)-CGP12177 binds at two beta(1)-adrenoceptor sites, human beta(1)-adrenoceptors, expressed in CHO cells, were labelled with (-)-[(3)H]-CGP12177. We compared the binding affinity and antagonist potency of 12 clinically used beta-blockers against the cyclic AMP-enhancing effects of (-)-isoprenaline and (-)-CGP12177.(-)-[(3)H]-CGP12177 bound to a high affinity site (H; K(H)=0.47 nM) and low affinity site (L); K(L)=235 nM). (-)-[(3)H]-CGP12177 dissociated from the beta(1)-adrenoceptors with a fast component (k(off)=0.45 min(-1)), consistent with the L-site, and a slow component (k(off)=0.017-0.033 min(-1)), consistent with the H-site. (-)-Isoprenaline and (-)-CGP12177 caused 96-fold and 12-fold maximal increases in cyclic AMP levels with -logEC(50)M of 8.2 and 7.6. (-)-CGP12177 antagonised the effects of (-)-isoprenaline with a pK(B) of 9.9. The beta-blockers antagonised the effects of (-)-isoprenaline more than the effects of (-)-CGP12177 with potency ratios: (-)-atenolol 1,000, (+/-)-metropolol 676, (-)-pindolol 631, (-)-timolol 589, (+/-)-carvedilol 204, (+/-)-oxprenolol 138, (+/-)-sotalol 132, (-)-propranolol 120, (+/-)-bisoprolol 95, (+/-)-alprenolol 81, (+/-)-nadolol 68 and (-)-bupranolol 56. In intact cells the binding constants of beta-blockers, estimated from competition with 3-5 nM (-)-[(3)H]-CGP12177 (binding to the H-site), correlated with the corresponding affinities estimated from antagonism of the (-)-isoprenaline effects. We conclude that (-)-[(3)H]-CGP12177 binds at two sites in the recombinant beta(1)-adrenoceptor. (-)-CGP12177 is an antagonist of catecholamine effects through the H-site and a non-conventional partial agonist through the L-site. beta-blockers are more potent antagonists through the H-site than the L-site.

Intrinsic sympathomimetic activity of (-)-pindolol mediated through a (-)-propranolol-resistant site of the beta1-adrenoceptor in human atrium and recombinant receptors.

The beta-blocker (-)-pindolol produces intrinsic sympathomimetic activity manifested clinically by cardiostimulation, but the beta-adrenoceptor subtype, which mediates these effects, is unknown. Recent work indicates the existence of a (-)-propranolol-resistant site of the cardiac beta(1)-adrenoceptor and we propose that it mediates the cardiostimulation evoked by (-)-pindolol. We compared the interaction of (-)-pindolol both with human atrial myocardium and with recombinant beta(1)-adrenoceptors. The effects of (-)-pindolol on paced human atrial trabeculae were studied in the presence of 3-isobutyl-1-methylxanthine (IBMX; 20 microM). (-)-Pindolol caused small negative and positive inotropic effects at nanomolar and micromolar concentrations respectively, which were unaffected by N(G)-monomethyl-L-arginine (L-NMMA, 10 microM), inconsistent with an involvement of nitric oxide. (-)-Pindolol, in the presence of (-)-propranolol, increased atrial contractile force and cAMP through recombinant beta(1)-adrenoceptors with identical potency (-logEC(50)M=6.5). The positive inotropic effects of (-)-pindolol were resistant to blockade by L-748,337 (100 nM), a beta(3)-adrenoceptor antagonist. (-)-CGP12177, known to act through the (-)-propranolol-resistant site of the beta(1)-adrenoceptor, also increased with similar potency atrial contractile force (-logEC(50)M=7.6) and cAMP at recombinant beta(1)-adrenoceptors (-logEC(50)M=7.7). (-)-Pindolol blocked the effects of (-)-CGP12177 in human atrium and recombinant beta(1)-adrenoceptors with similar equilibrium dissociation constants (pK(B)=6.5 and 6.3). Thus, stimulant potency and blocking potency of (-)-pindolol against (-)-CGP12177 agree. In contrast, (-)-pindolol was 200-400 times more effective at blocking the effects of a catecholamine than the effects of (-)-CGP12177 in both human atrium (pK(B)=9.1) and at recombinant beta(1)-adrenoceptors (pK(B)=8.6). We conclude that the cardiostimulant effects of (-)-pindolol in human atrial myocardium are mediated through a (-)-propranolol-resistant site of the beta(1)-adrenoceptor with low affinity for (-)-pindolol. In contrast, (-)-pindolol blocks the effects of catecholamines through a high-affinity site of the beta(1)-adrenoceptor. beta(3)-Adrenoceptors are not involved in the atrial effects of (-)-pindolol.

Physical and transcript map of the hereditary prostate cancer region at xq27.

We have recently mapped a locus for hereditary prostate cancer (termed HPCX) to the long arm of the X chromosome (Xq25-q27) through a genome-wide linkage study. Here we report the construction of an approximately 9-Mb sequence-ready bacterial clone contig map of Xq26.3-q27.3. The contig was constructed by screening BAC/PAC libraries with markers spaced at approximately 85-kb intervals. We identified overlapping clones by end-sequencing framework clones to generate 407 new sequence-tagged sites, followed by PCR verification of overlaps. Contig assembly was based on clone restriction fingerprinting and the landmark information. We identified a minimal overlap contig for genomic sequencing, which has yielded 7.7 Mb of finished sequence and 1.5 Mb of draft sequence. The transcriptional mapping effort localized 57 known and predicted genes by database searching, STS content mapping, and sequencing, followed by sequence annotation. These transcriptional units represent candidate genes for HPCX and multiple other hereditary diseases at Xq26.3-q27.3.