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`4
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`Bylund
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`dawned with the crystallization of the receptor concept and closed with the
`crystallization and determination of the three-dimensional structure of rhodop(cid:173)
`sin. At the start of the century, epinephrine and norepinephrine had not yet been
`isolated; by its end, knockout mice were available for all nine adrenergic receptor
`subtypes, and clinically relevant polymorphisms were being elucidated. This
`progress was the result of the hard work and insightful thinking of a remarkable
`cadre of investigators throughout the world. A few of these are mentioned in this
`chapter, although most (unfortunately) remain unnamed.
`A summary listing by decade of some of the more important developments in
`our understanding of adrenergic receptors is given in Table I. The accomplish(cid:173)
`ments are indeed impressive. I have chosen to divide the century into four major,
`overlapping epochs or eras, each named to represent the dominant focus of a
`given time period. The century started with the biochemical era, which lasted
`until the mid-l 960s and resulted in the isolation of many small compounds, such
`as norepinephrine, epinephrine, and the second messenger cyclic adenosine 5' -
`monophosphate (AMP). This was followed by what I term the physiological era,
`from about l 960totheearly 1980s; it was characterized by elegant use of isolated
`tissue preparations to elucidate the general characteristics of adrenergic recep(cid:173)
`tors. The pharmacological era, lasting from the mid- l 970s to the early 1990s,
`included not only the use the physiological techniques of the previous era, but
`also, perhaps more important, radioligand-bindingtechniques to classify, local(cid:173)
`ize, and characterize the types and subtypes of adrenergic receptors based on
`their interactions with a rich variety of agonists and antagonists. The molecular
`era, which is just now ending, started in the mid- l 980s and has seen the charac(cid:173)
`terization of receptors by the molecular biological techniques of cloning, site(cid:173)
`directed mutagenesis, and genetic engineering.
`
`2. The Biochemical Era (1901-1960)
`"Tentatively the first kind of receptor has been called the alpha adrenotropic
`receptor and the second kind the beta receptor." (Ahlquist, 1948)
`John Jacob Abel, a newly appointed professor of pharmacology at Johns
`Hopkins in Baltimore, Maryland, started work on the isolation of epinephrine
`about 1896 and by 1901 had a relatively pure preparation. He is generally cred(cid:173)
`ited with isolating the first hormone ( 1 ). Starting in the 1920s, Cannon attempted
`to identify the chemical transmitter of the sympathetic nervous system (which he
`called sympathin) and mistakenly concluded in 1933 that there were two sym·
`pathins, sympathin E (excitatory) and sympathin I (inhibitory) (2). This was
`partly because he was using a natural preparation, adrenaline, which at that time
`was a variable mixture of epinephrine and norepinephrine. It was not until the late
`1940s that von Euler finally established that norepinephrine was the predomin
`postganglionic neurotransmitter of the sympathetic nervous system ( 3 ).
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`8
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`Bylund
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`norepinephrine, whereas the P2-adrenergic receptor, responsible for relaxation
`of vascular, uterine, and airway smooth muscle, was less sensitive to norepineph(cid:173)
`rine compared to epinephrine. At the same time, Furchgott also proposed the
`presence of various types of P-adrenergic receptors ( 13 ). These studies deter(cid:173)
`mined the dissociation constant for an ex- (phentolamine) and P- (pronethalol)
`adrenergic receptor blocker in several isolated tissues. "The results will, I
`believe, ... show that there may be various types of beta receptors within the
`general class of such receptors."
`In the mid-1950s, Brown and Gillespie measured the levels of sympathin
`(norepinephrine) in the venous blood from the cat spleen following nerve stimu(cid:173)
`lation. In the presence of ex-adrenergic antagonists ( dibenamine or dibenzy line),
`the amount of sympathin released was greatly increased (14). This study was
`misinterpreted at that time as showing that the adrenergic receptor served as an
`important site in the loss of norepinephrine. In the early 1970s, based on studies
`in several laboratories utilizing tritiated norepinephrine, both Langer and Starke
`and coworkers correctly concluded that these drugs increased the output of nor(cid:173)
`epinephrine elicited by nerve stimulation (15,16). Starke then quickly showed
`that phenylephrine caused a dose-dependent inhibition of norepinephrine release
`from the isolated rabbit heart, leading to the conclusion of presynaptic regulation
`of norepinephrine release mediated by ex-adrenergic receptors ( 17 ). A few months
`later, Langer formalized the concept ( 18 ): "These presynaptic receptors would
`regulate the transmitter released by nerve stimulation. A negative feedback in(cid:173)
`hibition can be envisaged in which noradrenaline released by nerve stimulation
`would itself inhibit further release once a threshold concentration of the transmit(cid:173)
`ter is achieved in the neighborhood of the nerve ending. Block of these receptors
`would lead to an increase in release of the transmitter by nerve stimulation."
`This work in tum led to a proposal for the subclassification of the ~adrenergic
`receptors based on their anatomical location ( 19 ). "These results are compatible
`with the view that the pre- and the post-synaptic ex receptors are not identical.
`Perhaps the postsynaptic ex receptor that mediates the response of the effector
`organ should be referred to as ex-1 , while the presynaptic ex receptor that regulates
`transmitter release should be called ex-2." It was, however, quickly realized that
`not all ex-receptors with exi pharmacological characteristics were presynaptic.
`Thus, a functional classification of ex-adrenergic receptors was proposed with the
`excitatory receptors as ex1 and the inhibitory receptors as <Xi (20 ). It was noted,
`however, that there was a different agonist potency order for the two subtypes.
`Although the functional aspect of their proposal turned out not to be generally
`applicable, the pharmacological definition based on the rank order of potency of
`agonists has prevailed.
`It also became apparent that not all of the P-adrenergic receptor-mediated
`responses could be classified as either p1 or p2, suggesting the existence of at least
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`10
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`Bylund
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`Fig. 1. A cartoon from 1988 indicating the frustration some investigators felt at the
`seemingly endless proliferation of adrenergic receptor subtypes. (From ref. 40; © 1988,
`with permission from Elsevier.)
`
`radioligand-binding studies showing that the two subtypes had differential sen(cid:173)
`sitivities to the site-directed alkylating agent chloroethylclonidine ( 33 ).
`The evidence for exi-receptor subtypes came from binding studies in various
`tissues and cell lines ( 34 ). The IXiA and «Xia subtypes were initially defined based
`on their differential affinities for adrenergic agents such as prazosin and
`oxymetazoline (35), and their existence was confirmed by functional studies
`( 36 ). The third subtype, «Xie. was identified originally in an opossum kidney cell
`line using radioligand-binding studies (37). A fourth pharmacological subtype
`the Uio. was identified in the rat and cow ( 38,39 ). Subsequently, it was shown th
`this pharmacological subtype was a species orthologue of the human UiA su
`type, and thus it is not considered a separate genetic subtype.
`During the second half of the 1980s, when new receptor subtypes were pf
`posed regularly, there was considerable opposition to this seemingly endle
`proliferation of subtypes. One reviewer chided the author on this point in
`review of one of his papers in 1987: "Shall we expect proposals for furt
`equally trivial revisions each time a new ligand is found with very high selectiv
`ity between receptors for which all previously known ligands had only m
`selectivity?" Perhaps the mood at this time is accurately reflected in the cart
`(Fig. l) from my 1988 review of a-adrenergic receptor classification ( 40 ). In
`cartoon, it should be noted that the investigator is trying to push the "subty
`back into the hat, or at least prevent them from popping out, rather than hap ·
`pulling them out.
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`12
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`Bylund
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`rhodopsin was subsequently cloned and also found to have seven putative mem(cid:173)
`brane-spanning regions ( 47). In 1986, Dixon, Strader, Lefkowitz, and colleagues
`cloned the ~2-adrenergic receptor using olignucleotide probes based on the
`sequence of a CNBr peptide obtained from the ~2-receptor purified to homoge(cid:173)
`neity from hamster lung (48). They noted that its predicted amino acid sequence
`had significant homology with bovine rhodopsin and suggested that, like the
`rhodopsins, the ~2-adrenergic receptor possessed multiple membrane-spanning
`regions, later shown to be seven. It is now recognized that adrenergic receptors
`are seven TM receptors, which consist of a single polypeptide chain with seven
`hydrophobic regions that are thought to form a-helical structures and span or
`transverse the membrane.
`Lefkowitz and colleagues quickly realized the potential existence of a large
`family of these seven TM receptors and started cloning other receptors by
`homology screening. Using the human ~2-adrenergic receptor as a probe, they
`isolated a genomic clone called G-21 , the first of the "orphan" receptors (49),
`which was subsequently shown to be the 5-HT1A receptor (50). This clone in
`tum was used as a probe to clone the human ~1 -adrenergic receptor (51 ). The
`~3-receptor, which had been pharmacologically defined in 1984 (21 ), was
`cloned in 1989 by Strosberg's lab (52). A 134-receptor has also been postulated
`and was even "canonized" by the Adrenergic Receptor Subcommittee of the
`IUPHAR Committee on Receptor Nomenclature and Drug Classification in
`1998 (53). It has not been cloned, however, and thus definitive evidence of its
`existence is lacking. The putative 134-receptor is now thought to be a "state" of
`the 131-adrenergic receptor (54 ).
`The CX:zA-receptor from the human platelet was cloned in 1987 by Lefkowitz's
`group based on the same strategy they used for the 132-adrenergic receptor (55).
`The gene was found to be located on chromosome 10, and thus it was subse(cid:173)
`quently called a:z-CIO. A Southern blot of human genomic deoxyribonucleic
`acid (DNA) blotted with a probe from this receptor indicated the existence of
`genes homologous to CX:zA receptor on chromosomes 2 and 4 and suggested that
`these might represent other pharmacologically defined a:z-receptor subtypes.
`A second subtype was cloned two years later from a human kidney comple·
`mentary DNA (cDNA) library using the gene for the human CX:zA-adrenergic
`receptor as a probe (56). The gene for this subtype was shown to be located OD
`human chromosome 4 and thus became known as exi-C4; it was initially identi·
`fled as the a:z8-pharmacological subtype, although this was later shown to be ill
`error.
`A third exi-subtype was cloned first from a rat kidney cDNA library scree
`with an oligonucleotide complementary to a highly conserved region found in
`biogenic amine receptors that had been described up to that time (57). It w
`called the a:z-RNG (for rat nonglycosylated) and was correctly identified as
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`14
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`Bylund
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`which allows for the alteration or deletion of specific amino acids in a receptor
`(see Chapter 2). The first application of this technique, which was then used by
`many workers to define ligand-binding site and signaling mechanism, was by the
`Strader lab in 1987 ( 69 ). Based on a series of deletion mutants of the hamster p2-
`adrenergic receptor that showed that most of the hydrophilic residues are not
`directly involved in ligand binding, they concluded that the binding site must
`involve residues within the hydrophobic TM domain. They then went on to show
`the importance of Asp 113 in TM III in agonist binding to the receptor (70) and
`of the serine residues 204 and 207 in TM Vin the activation of the p2 receptor (71 ).
`The third extremely useful technique of molecular biology was the use of
`transgenic animals, particularly gene targeting to disrupt the expression of a spe(cid:173)
`cific receptor to generate so-called knockout mice (see Chapters 8-11). Interest(cid:173)
`ingly, the first adrenergic receptor to be knocked out was the P3-receptor in 1995
`by Lowell's lab (72). These mice had only modestly increased lipid stores but
`lacked the physiological responses to administered P3-agonists observed in wild(cid:173)
`type mice. The next year, Kobilka's lab generated mice lacking the p,-adrenergic
`receptor (73). In addition to developmental defects, these mice lacked both chro(cid:173)
`notropic and inotropic responses to administered P-agonists. The P2-knockout
`mice, generated several years later by Kobilka, showed that this subtype primarily
`influences the smooth muscle relaxant properties of several tissues (74). Simple
`breeding experiments have allowed for the generation of the three combinations of
`double P-receptor knockouts, as well as the triple knockout (75) (see Chapter 10).
`Mice lacking the a 18-adrenergic receptor were generated by the Cotecchia lab
`in 1997 (76 ). Whereas basal blood pressure was not altered in these animals, the
`hypertensive response to a 1-agonists was significantly blunted. Subsequently,
`the a,A-(77) and am-knockouts (78) were generated. All three a,-subtypes play
`important roles in the cardiovascular system, and the a 18 may be particularly
`important in the central nervous system (reviewed in ref. 79) (see Chapter 8).
`In 1996, the Limbird lab genetically modified the <XiA-receptor by the hit-and(cid:173)
`run technique, which essentially is site-specific mutagenesis in vivo (80) (see
`Chapter 9). Mice homozygous for the D79N substitution lack the profound hypo(cid:173)
`tension normally induced by exi-agonists. Concurrently, the Kobilka lab produced
`<Xie- and <Xie-knockouts (81) and then a few years later the <XiA-knockout (82).
`These studies indicated that primarily the <XiA-subtype mediates most of the clas·
`sical exi-adrenergic functions, such as hypotension, sedation, analgesia, and hypo(cid:173)
`thermia. The exi8-subtype is the principal mediator of the hypertensive response
`to exi-agonists, whereas the <Xie-subtype appears to be involved in many central
`nervous system functions (reviewed in ref. 83).
`The fourth molecular technique of note to be applied to the adrenergic rece
`tors was the determination of receptor polymorphisms in human populatio
`Polymorphisms are frequently occurring genetic variants (by contrast with
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`16
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`Bylund
`
`as we take what we are learning about these magnificent receptors and more fully
`integrate that knowledge into better understanding of how they function in the
`whole animal in relation to other neurohormonal systems as components of
`various physiological systems. This in turn, we all expect, will lead to better
`therapeutics as we more rationally use existing drugs for selected populations
`and develop new drugs with greater selectivity, efficacy, and fewer side effects.
`
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`18
`
`Bylund
`
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`
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`20
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`Bylund
`
`71 . Strader CD, Candelore MR, Hill WS, Sigal IS , Dixon RA. Identification of two
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`
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`Exhibit 1032-22
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