Roger Summers and Bronwyn Evans, Department of Pharmacology, Monash University, Clayton, Vic 3800, Australia

The classical b₁- and b₂-adrenoceptors (ARs) are important mediators of responses to noradrenaline released from sympathetic nerve terminals and to circulating adrenaline. The b-adrenoceptors are distinguished from a-adrenoceptors pharmacologically and by amino acid sequence. They are stimulated by (-) isoprenaline and blocked by (-) propranolol, and the development of selective compounds has produced valuable drugs for the treatment of hypertension, cardiac arrhythmias, ischaemic heart disease and asthma, and for prophylaxis of myocardial infarction. The observation of propranolol-resistant responses to isoprenaline and sympathetic stimulation, including lipolysis in adipose tissues and relaxation of gastrointestinal smooth muscle (1, 2) suggested another b-adrenoceptor subtype, confirmed by cloning and expression of the b3-AR gene . The b3-AR is resistant to propranolol and selectively stimulated by lipolytic agonists such as BRL 37344 and CL 316243 . It is also stimulated by CGP 12177, which under most circumstances acts as an antagonist at b₁- and b₂-ARs. Selective agonists and antagonists now exist for all three b-adrenoceptors.

 

b-Adrenoceptors are Class 1 G protein-coupled receptors (GPCRs) with a characteristic 7 transmembrane domain structure. mRNA and protein for the b₁-AR is found in brain, heart, kidney and adipose tissue and for the b₂-AR in brain, lung, trachea, uterus and human heart, while b3-AR mRNA is restricted to brown (BAT) and white adipose tissue (WAT), gallbladder, stomach, colon, ileum, prostate gland and brain (5,6,7). The genes encoding the 3 known b-adrenoceptors show a clear ancestral relationship, with regions of identity in nucleotide as well as amino acid sequence. However the b3-AR gene contains three exons and two introns, differing from the b1- and b2-AR genes which are intronless (8,9). Exon 1 of the b3-AR gene encodes 390 amino acids including the 7 transmembrane domains, exon 2 encodes the last 13 amino acids of the C-terminal tail, and exon 3 includes only the 3'-untranslated region.

 

There is redundancy in the response mediated by b3-ARs and classical b1/b2-ARs in some cell types. For example, b₁-, b₂-, and b3-ARs all mediate lipolysis in adipocytes and their relative contributions depend on the species, the type of fat depot studied, and other physiological influences . Stimulation of any of the 3 b-ARs increases cAMP production via activation of G_s, and in most situations the effects of b-AR agonists can be mimicked by cAMP analogues or forskolin . There is evidence that b₃-ARs also interact with G_i , and in some cell types although the predominant receptor present is the b₃-AR, increases in cAMP occur primarily through b₁- and b₂-ARs . Recent work demonstrates that b₃-AR also signal through the MAP kinase (ERK 1/2) pathway. There are further distinctions between the b₃-AR and classical b₁- and b₂-ARs, including their regulatory properties. At the protein level, desensitization of b₁- and b₂-AR responses upon agonist stimulation involves increased phosphorylation of occupied receptors, uncoupling and inactivation. b₁- and b₂-ARs have serine and threonine residues in the C-terminal tail which act as substrates for G protein-coupled receptor kinases, and consensus sequences for phosphorylation by cAMP-dependent protein kinase (PKA). The b₃-AR lacks a PKA phosphorylation site, and has fewer serine and threonine residues in the C-terminal tail (Fig 1). Experimentally, the b3-AR is resistant to short term agonist-promoted desensitization. Studies on chimeric b₂/b3-ARs show that domains within the C-terminal tail and second and third intracellular loops which are present in the b₂-AR are the major determinants of desensitisation (12,13).

b-adrenoceptor polymorphisms

This rapidly developing area studies frequently occurring variants of b-AR that may be important in determining the response to drugs and in causation of disease. A number of polymorphisms have been described for the b₂-AR including Arg16Gly, Gln27Glu, Val34Met and Thr164Ile. Arg16Gly is associated with increased down regulation whereas Gln27Glu shows decreased down regulation . Thr164Ile is associated with defects in signalling and function in the heart. There is an emerging case that these receptor polymorphisms may be associated with the aetiology of asthma, hypertension and obesity. A polymorphism of the b₃-AR the Trp64Arg was originally discovered in the Pima Indians of North America that have a high incidence of the mutation in association with late onset diabetes and obesity. The mutant b₃-AR has less efficient signal transduction properties than the Arg 64 receptor. The polymorphism is now known to be widely distributed throughout many ethnic groups but more work is needed to finally establish the connection with obesity and diabetes.

 

The mouse b3-AR splice variant

We have recently demonstrated alternative splicing of the mouse b3-AR transcript at the junction between exons 1 and 2 . Use of an alternative acceptor site 100 bp upstream of the usual start of exon 2 results in the production of mRNA encoding a b3-AR variant (b3B-AR). mRNA coding for this receptor represents 7.6% of the b3-AR transcripts in BAT, 21% in WAT and 16% in ileum smooth muscle. The highest proportion of b3B-AR transcripts occurs in brain, with 38% in hypothalamus and 32% in cortex. We have subcloned the complete coding regions of the b3A-AR and b3B-AR mRNAs into the mammalian expression vector pcDNA3.1+ (Invitrogen) and transfected CHO-K1 cells to produce stable transformants selected using G418 (neomycin). Comparison of two clonal cell lines with similar levels of b3-AR mRNA and binding using a cytosensor microphysiometer demonstrate that both receptors are functional, being stimulated by selective b3-AR agonists and blocked by the selective b3-AR antagonist SR 59230A. However, maximal responses to a wide range of agonists were greater for the b_3a- compared to the b_3b-AR In competition binding studies, the pharmacological properties of the receptors were found to be identical.

 

The 'b4-adrenoceptor'

There is mounting evidence for the presence of additional putative b-adrenoceptor subtypes. Attention has focused on the putative b4-AR found in mammalian heart and adipose tissue. The inotropic response to CGP 12177 in human, mouse and rat atria is maintained in b₃-AR knockout mice (16,17). Similarly, BAT oxygen consumption is stimulated maximally by CGP 12177 in both wild-type and b₃-AR knockout mice. CGP 12177-stimulated lipolysis in rat white adipocytes is only partially blocked by the b3-AR selective antagonist SR 59230A, suggesting participation by another b-adrenoceptor subtype. Although these studies demonstrate that putative b4-ARs cannot be accounted for by b3-ARs, they do not conclusively rule out b₁- /b₂-ARs. In fact CGP 12177 acts as a partial agonist at human b₁-ARs but not b₂-ARs expressed in CHO and CHW cell lines . Thus there are several possible explanations for the 'b4-AR', namely (i) it is a novel b-adrenoceptor related to the 3 known b-ARs, (ii) it represents a form of the b₁-AR, (iii) it is a dimer of a known b-AR with altered pharmacological properties or (iv) a combination of a known receptor with a RAMP protein. There is also a novel b-adrenoceptor cloned from turkey that has been called the b4-AR . This receptor is distinct from the turkey b₁- and b₂-ARs, that show a surprisingly high homology with their mammalian counterparts. The turkey b4-AR may be a homologue of the mammalian b4-AR or the b3-AR, or may be unique to avian species.

 

The skeletal muscle 'b-adrenoceptor'

In animal models of genetic and diet-induced obesity, b3-AR selective agonists promote weight loss and improve insulin sensitivity , while targeted disruption of the b3-AR gene in mice causes mild obesity and doubling of lipid stores . Agonists selective at the b3-AR are being developed as agents for the treatment of human obesity. A human b3-AR agonist L-755,507 produces acute increases in metabolic rate in rhesus monkeys . Long-term treatment of obese rats and mice with CL 316243 produces weight loss via stimulation of lipolysis in WAT and thermogenesis in BAT. However, several studies suggest that b3-AR selective agonists also act on skeletal muscle in addition to WAT and BAT. For instance, chronic treatment produces elevated thermogenesis in muscle tissues , and consequently increased formation of mitochondria and induction of the genes encoding mitochondrial uncoupling proteins (UCP-1 and 3). Also, in vivo and in vitro studies show that acute treatment with BRL 37344 or CL 316243 increases glucose uptake by muscles and cultured L6 myocytes by an unknown mechanism. There is negligible b1-AR or b3-AR mRNA in rat soleus muscle or L6 myocytes yet BRL 37344 and CL 316243 increase glucose uptake at low concentrations. BRL 37344 acts as a weak agonist at b2-ARs, but activation of this subtype causes a decrease in glucose uptake . Finally, the soleus muscle b-AR is highly sensitive to BRL 37344 whereas the 'putative b4-AR' is unresponsive to this agonist. It remains to be established how b₃-AR agonists enhance glucose uptake in skeletal muscle and whether this effect is mediated via a novel b-adrenoceptor subtype.

 

Conclusions

The b₂-AR was the first amine responsive G-protein coupled receptor to be cloned. In the 12 years since, enormous strides have been made in our understanding of b-adrenoceptor subtypes and their signal transduction mechanisms. However it is clear that much remains to be done particularly in the field of novel receptors related to b-AR, new roles for the receptors and in establishing the significance of receptor polymorphisms for drug action and disease.

 

Supported by the National Health and medical Research Council of Australia.

 

Contact: roger.summers @med.monash.edu.au or bronwyn.evans@med.monash.edu.au