Stuart A. Green

A Gain-of-function Polymorphism in a G-protein Coupling Domain of the Human β1-Adrenergic Receptor

The β1-adrenergic receptor (β1AR) is a key cell surface signaling protein expressed in the heart and other organs that mediates the actions of catecholamines of the sympathetic nervous system. A polymorphism in the intracellular cytoplasmic tail near the seventh transmembrane-spanning segment of the human β1AR has been identified in a cohort of normal individuals. At amino acid position 389, Gly or Arg can be found (allele frequencies 0.26 and 0.74, respectively), the former previously considered as the human wild-type β1AR. Using site-directed mutagenesis to mimic the two variants, CHW-1102 cells were permanently transfected to express the Gly-389 and Arg-389 receptors. In functional studies with matched expression, the Arg-389 receptors had slightly higher basal levels of adenylyl cyclase activities (10.7 ± 1.2 versus 6.1 ± 0.4 pmol/min/mg). However, maximal isoproterenol-stimulated levels weremarkedly higher for the Arg-389 as compared to the Gly-389 receptor (63.3 ± 6.1 versus 20.9 ± 2.0 pmol/min/mg). Agonist-promoted [35S]guanosine 5′-O-(thiotriphosphate) binding was also increased with the Arg-389 receptor consistent with enhanced coupling to Gsand increased adenylyl cyclase activation. In agonist competition studies carried out in the absence of guanosine 5′-(β,γ-imido)triphosphate, high affinity binding could not be resolved with the Gly-389 receptor, whereas Arg-389 displayed an accumulation of the agonist high affinity receptor complex (R H = 26%). Taken together, these data indicate that this polymorphic variation of the human β1AR results in alterations of receptor-Gs interaction with functional signal transduction consequences, consistent with its localization in a putative G-protein binding domain. The genetic variation of β1AR at this locus may be the basis of interindividual differences in pathophysiologic characteristics or in the response to therapeutic βAR agonists and antagonists in cardiovascular and other diseases.

cAMP-Dependent Regulation of Cardiac L-Type Ca2+ Channels Requires Membrane Targeting of PKA and Phosphorylation of Channel Subunits

The cardiac L-type Ca2+ channel is a textbook example of an ion channel regulated by protein phosphorylation; however, the molecular events that underlie its regulation remain unknown. Here, we report that in transiently transfected HEK293 cells expressing L-type channels, elevations in cAMP resulted in phosphorylation of the alpha1C and beta2a channel subunits and increases in channel activity. Channel phosphorylation and regulation were facilitated by submembrane targeting of protein kinase A (PKA), through association with an A-kinase anchoring protein called AKAP79. In transfected cells expressing a mutant AKAP79 that is unable to bind PKA, phosphorylation of the alpha1C subunit and regulation of channel activity were not observed. Furthermore, we have demonstrated that the association of an AKAP with PKA was required for beta-adrenergic receptor-mediated regulation of L-type channels in native cardiac myocytes, illustrating that the events observed in the heterologous expression system reflect those occurring in the native system. Mutation of Ser1928 to alanine in the C-terminus of the alpha1C subunit resulted in a complete loss of cAMP-mediated phosphorylation and a loss of channel regulation. Thus, the PKA-mediated regulation of L-type Ca2+ channels is critically dependent on a functional AKAP and phosphorylation of the alpha1C subunit at Ser1928.

Genetic polymorphisms of the beta 2-adrenergic receptor in nocturnal and nonnocturnal asthma. Evidence that Gly16 correlates with the nocturnal phenotype.

Nocturnal asthma represents a unique subset of patients with asthma who experience worsening symptoms and airflow obstruction at night. The basis for this phenotype of asthma is not known, but beta 2-adrenergic receptors (beta 2AR) are known to downregulate overnight in nocturnal asthmatics but not normal subjects or nonnocturnal asthmatics. We have recently delineated three polymorphic loci within the coding block of the beta 2AR which alter amino acids at positions 16, 27, and 164 and impart specific biochemical and pharmacologic phenotypes to the receptor. In site-directed mutagenesis/recombinant expression studies we have found that glycine at position 16 (Gly16) imparts an accelerated agonist-promoted downregulation of beta 2AR as compared to arginine at this position (Arg16). We hypothesized that Gly16 might be overrepresented in nocturnal asthmatics and thus determined the beta 2AR genotypes of two well-defined asthmatic cohorts: 23 nocturnal asthmatics with 34 +/- 2% nocturnal depression of peak expiratory flow rates, and 22 nonnocturnal asthmatics with virtually no such depression (2.3 +/- 0.8%). The frequency of the Gly16 allele was 80.4% in the nocturnal group as compared to 52.2% in the nonnocturnal group, while the Arg16 allele was present in 19.6 and 47.8%, respectively. This overrepresentation of the Gly16 allele in nocturnal asthma was significant at P = 0.007 with an odds ratio of having nocturnal asthma and the Gly16 polymorphism being 3.8. Comparisons of the two cohorts as to homozygosity for Gly16, homozygosity for Arg16, or heterozygosity were also consistent with segregation of Gly16 with nocturnal asthma. There was no difference in the frequency of polymorphisms at loci 27 (Gln27 or Glu27) and 164 (Thr164 or Ile164) between the two groups. Thus the Gly16 polymorphism of the beta 2AR, which imparts an enhanced downregulation of receptor number, is overrepresented in nocturnal asthma and appears to be an important genetic factor in the expression of this asthmatic phenotype.

The Ile164 β2-adrenoceptor polymorphism alters salmeterol exosite binding and conventional agonist coupling to Gs

beta(2)-adrenoceptors (beta(2)AR) are polymorphic at amino acid 164 (Thr or Ile) of the fourth transmembrane domain. In transfected fibroblasts, six agonists commonly used in the treatment of bronchospasm were studied. Isoproterenol, albuterol, metaproterenol, terbutaline, formoterol, and salmeterol displayed decreased binding affinities (K(i)s were 1.2-3.0-fold higher) and a significant degree of impaired maximal stimulation of adenylyl cyclase ( approximately 40%), was observed with all agonists for the Ile164 receptor. The ratios of signal transduction efficiencies (Tau function, Ile164/Thr164) varied from a low of 0.17 for terbutaline to 0.49 for salmeterol. In addition, Ile164 bound salmeterol at the exosite, as delineated in perfusion washout studies, at a decreased level (31+/-4.8% vs. 49+/-4.4% retained salmeterol, respectively, P=0.02). In cAMP production studies under perfusion conditions, this decreased exosite binding caused a approximately 50% decrease in the duration of action of salmeterol at Ile164 (t(1/2)=21.0+/-3.6 vs. 46.8+/-4.1 min for Thr164, P=0.001). The durations of action for isoproterenol and formoterol under similar perfusion conditions were not different between the two receptors. These in vitro results indicate the Ile164 polymorphic receptor represents a pharmacogenetic locus for the most commonly utilized agonists in the treatment of asthma with a unique phenotype for salmeterol.

Coupling of β-Adrenergic Receptors to Cardiac L-Type Ca2+ Channels: Preferential Coupling of the β1 Versus β2 Receptor Subtype and Evidence for PKA-Independent Activation of the Channel

Beta1- and beta2-adrenergic receptors (beta-ARs) co-exist in mammalian heart, and it is generally accepted that both activate adenylyl cyclase (AC), resulting in increased levels of cAMP and subsequent activation of L-type Ca2+ channels (CaCh). To investigate the contribution of each beta-AR subtype in AC and CaCh coupling, we stably expressed cardiac CaCh alpha1 and beta2 subunits along with either beta1-AR or beta2-AR in CHW fibroblasts. Co-expression of either beta-AR with CaCh subunits conferred responsiveness of AC and CaCh to isoproterenol (ISO), which was not observed in non-transfected cells. ISO-promoted cAMP formation occurred at a lower EC50 through the beta2-AR than through the beta1-AR (0.13 +/- 0.01 vs. 0.6 +/- 0.14 nM). In contrast, activation of CaCh was more efficacious via the beta1-AR than the beta2-AR (EC50 for CaCh activation = 238 +/- 33 vs. 1057 +/- 113 nM). Pre-treatment with pertussis toxin (PTX) had no effect upon the responsiveness of either cAMP formation or CaCh activation through either receptor. We conclude (1) that beta1-ARs exhibit preferential coupling to CaCh activation, versus that observed for the beta2-AR; (2) that this preferential coupling cannot be explained solely by cAMP-dependent processes; and (3) that the relative attenuation of beta2-AR-promoted CaCh activation is not due to receptor coupling to PTX-sensitive G proteins. Thus, it is likely that other subtype-specific, cAMP-independent coupling of the beta-AR to CaCh is present.

Role of βARK in Long-Term Agonist-Promoted Desensitisation of the β2-Adrenergic Receptor

Phosphorylation of the β2-adrenergic receptor (β2AR) is the initial event that underlies rapid agonist-promoted desensitisation. However, the role of phosphorylation in mediating long-term β2AR desensitisation is not known. To investigate this possibility, we performed intact cell phosphorylation studies with COS-7 cells transiently expressing an epitope tagged wild-type β2AR and found that receptor phosphorylation in cells treated with 1 μM isoproterenol for 24 h was ∼4-fold over the basal state. This finding suggested that persistent phosphorylation of the receptor might contribute to functional long-term desensitisation which we further explored with mutated β2AR lacking the determinants of phosphorylation by the βAR kinase (βARK), PKA or both. In CHW cells expressing the WT β2AR, pretreatment with 1 μM isoproterenol for 24 h reduced the isoproterenol-stimulated cAMP response by 82 ± 5%. Substitution of the PKA sites with alanines had no effect on the extent of desensitisation (77 ± 6%, P = NS compared to WT). In contrast, desensitisation was only 49 ± 4% (P < 0.001 compared to WT) when the βARK sites were similarly substituted. Removal of both the βARK and PKA sites impaired desensitisation to the same extent as the βARK mutant. The extent of receptor loss (downregulation) was the same among all of the cell lines used and therefore could not account for the observed differences in desensitisation. Cellular βARK activity, assessed by a rhodopsin phosphorylation assay, was equivalent in all cell lines and was unaffected by agonist treatment. PKA activity, however, was dynamically regulated, increasing 4-fold over basal levels after 15 min of isoproterenol and returning to near basal levels after 24 h. The lower level of PKA activity after long-term agonist exposure may therefore have contributed to the apparent lack of effect of removing PKA sites. Nonetheless, long-term desensitisation was clearly attenuated with β2AR lacking βARK phosphorylation sites. These findings show that in addition to its role in regulating short-term desensitisation, βARK-mediated phosphorylation is an important mechanism underlying long-term desensitisation of the β2AR as well.

Modulation of cardiac Ca2+channels by isoproterenol studied in transgenic mice with altered SR Ca2+ content

Phospholamban (PLB) ablation is associated with enhanced sarcoplasmic reticulum (SR) Ca2+ uptake and attenuation of the cardiac contractile responses to β-adrenergic agonists. In the present study, we compared the effects of isoproterenol (Iso) on the Ca2+ currents ( I Ca) of ventricular myocytes isolated from wild-type (WT) and PLB knockout (PLB-KO) mice. Current density and voltage dependence of I Ca were similar between WT and PLB-KO cells. However, I Ca recorded from PLB-KO myocytes had significantly faster decay kinetics. Iso increased I Ca amplitude in both groups in a dose-dependent manner (50% effective concentration, 57.1 nM). Iso did not alter the rate of I Ca inactivation in WT cells but significantly prolonged the rate of inactivation in PLB-KO cells. When Ba2+ was used as the charge carrier, Iso slowed the decay of the current in both WT and PLB-KO cells. Depletion of SR Ca2+ by ryanodine also slowed the rate of inactivation of I Ca, and subsequent application of Iso further reduced the inactivation rate of both groups. These results suggest that enhanced Ca2+ release from the SR offsets the slowing effects of β-adrenergic receptor stimulation on the rate of inactivation of I Ca.