Expression of mammalian cell-surface receptors in higher eukaryotic systems

Expression of mammalian cell-surface receptors in higher eukaryotic systems Michael I. Browne and Joanna M. Balcarek SmithKline Beecham Pharmaceuticals, Welwyn, UK and Upper Merion, USA

The study of recombinant receptors has progressed rapidly over the past year. These receptors are complex, often multimeric, protein systems. We have seen further progress in the use of mammalian cell lines, but two areas of development in receptor expression are notable: first, increasing use and success of the baculovirus system; and second, improved, rapid methods for detecting signal transduction.

Current Opinion in Biotechnology 1993, 4:553-557

Mammalian cell expression of cloned receptors

Introduction Human cell-surface receptors provide a key point for intervention in, and understanding of, numerous pathological states such as neurodegenerative disease, functional psychoses, hypertension, allergy and gastrointestinal disorders. While receptor-based research has a distinguished track-record of successful clinical application pre-dating the era of recombinant DNA, the increasingly rapid pace of progress in receptor biology is, in large part, a result of the use of molecular approaches. The structure of mammalian cell-surface receptors varies widely, as does the number of subunits that comprises them: the large seven transmembrane G protein linked class (TTM) receptor contains only one subunit, whereas the y-amino butyric acid (GABA) receptor consists of a number of subunits probably comprising a heteropentamer. The mechanism by which receptors interact with the cognate intracellular signalling system is also highly varied; for example, coupling can be mediated either by G proteins or b y gating of an ion channel (see [1] for a review). Each n e w recombinant receptor has the potential to challenge existing expression techniques. Although some successes in expressing mammalian receptors in lower organisms have b e e n reported [2,3], most mammalian receptors are expressed using higher eukaryotic systems. This review addresses recent advances in mammalian, Xenopus and insect receptor expression systems, highlighting both successes and problems.

Dopamine and serotonin receptors

The dopamine and serotonin (5HT) receptors are of key clinical importance - - dopamine is implicated in schizophrenia and Parkinsonism; 5HT is of importance in depression, anxiety, and migraine. They belong, with one exception (5HT3), to the 7TM class.

Until very recently, classical pharmacology had described only two dopamine (D) receptor subtypes: the D1 receptor, which stimulates adenylyl cyclase activity, and the D2 receptor, which inhibits adenylyl cyclase activity. With the advent of molecular biology, cloning has revealed a family of receptors with at least five human dopamine receptors ( h D l - h D 5 ) [4] and closely related rat homologues (rD). Both hD1 and hD5 (encoded b y intron-less genes) show a 'Dl-like' pharmacology, while the hD2, hD3 and hD4 (encoded by genes with introns) are more 'D2qike' in nature. It has proved relatively straightforward to express recombinant D1, D2 and D3 receptors (at levels of up to several pmole receptor/mg protein) in mammalian cells. Furthermore, hD1 expressed in COS cells (a transient expression system), mouse Ltl¢- cells and human embryonic kidney (HEK) 293 cells [5-7], hD5 expressed in rat somatomammatrophic GH4C1 [8] cells, and hD2 expressed in Chinese hamster ovary (CHO) [9,10] as well as HEK 293 cells [11], all show coupling with adenylyl cyclase in a m a n n e r that closely matches the pharmacological profile of D1 and D2 receptors. The D3 and D4 receptors have b e e n more problematical: demonstration of expressed hD3 functionality has proved elusive

Abbreviations CHO--Chinese hamster ovary; 5HT---serotonin; D---dopamine receptor; GABA--y-amino butyric acid; hD--human dopamine receptor; HEK--human embryonic kidney; IL--interleukin; PI--phosphatidyl inositol; rD--rat dopamine receptor. © Current Biology Ltd ISSN 0958-1669

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Expression systems [12,13] and even expression of the D4 receptors has been difficult. Initially, the hD4 receptor was obtained as a genomic clone and transient expression was attempted in COS7 cells; however, the expression obtained was too low to be useful [14"-]. Only after the cloning of a partial cDNA, which was used to form a hybrid gene/cDNA construct, was it possible to reach even 200-300 fmol receptor/mg protein. Thus far, it has not proved possible to stably express the hD4 receptor in CliO, Ltkor GH4C1 ceils at detectable levels (O Civelli, personal communication). The reason for the lack of stable expression of hD4 is unclear at present. Recently, it has b e e n shown that removal of the remaining introns in the hD4 gene/cDNA hybrid and replacement of silent GC base pairs with AT pairs (to reduce the unusually high GC content of the coding region) can improve expression in the COS system to 2pmole receptor/rag protein [15"q. It is not clear whether these manipulations contribute singly or in combination to improved expression. Characterization of the rD4 receptor has also proved problematical, with expression of the gene in the murine CCL 1.3 fibroblast cell line reportedly achieving levels of only 5 fmol receptor/mg protein [16]. Thus far, studies with a non-hydrolyzable GTP analogue do implicate hD4 in some form of coupling, although this has not b e e n confirmed in recombinant systems. Work on photoreceptors in the mouse retina, however, does show that native murine D4 receptor is able to inhibit adenylyl cyclase activity [17]. To date, 14 pharmacologically distinct subtypes of 5HT receptor have b e e n identified in mammals. These subtypes are divided into five subfamilies (5HT1-5HT5) on the basis of pharmacology and gene structure [18-32].

like pKi values. Although the presence of high-affinity agonist binding would seem to indicate some form of effector coupling, no PI- or cyclase-mediated signal transduction is detected [25], suggesting that the 5HT5A receptor cannot couple efficiently to the Gprotein repertoire (Gs, Gi and Gq) expressed in NIH3T3 cells. Indeed, Go and Gx, which predominate in neurons, are not found in many commonly used fibroblastic cell lines, and attention is turning to more highly specialized lines in an attempt to circumvent this problem. We must await the outcome of transfection studies utilizing other neural cell lines to discover whether the 5HT5A receptors, and the more recently cloned 5HT5B receptors, do indeed require different G proteins a n d / o r interact with effector systems other than adenylyl cyclase or PI (e.g. ion channels). A recent study by Adham et al. [33"] on overexpression of the 5HT1B receptor sounds yet another cautionary note in the area of effector coupling and functional response. High-level receptor expression (> I pmol receptor/rag protein) is often considered desirable; however, it n o w appears that overly high expression can alter the intrinsic activity of agonists and antagonists. Using mouse Y-1 cells expressing high levels (8 pmol receptor/mg protein) of the rat 5HTIB receptor, Adham et al. [33"] have observed that the ECs0 values for agonists are lower than expected from measured Ki values. They also note that ~-adrenergic antagonists, reported elsewhere to b e partial agonists or antagonists at this site, act as full agonists [33"]. By using an irreversible receptor antagonist, these discrepancies are shown to result, at least in part, from the large receptor reserve, which exists in this cell line. Receptor density-dependent changes in the intrinsic activity of drugs (from full agonist to silent antagonist) have also been reported for the 5HTIA receptor transfected into HeLa ceils [34]. It is unlikely, however, that receptor number is the only determinant of intrinsic activity; it has b e e n shown, for example, that compounds which are potent antagonists of the 5HTIB receptor in brain ceils are full agonists in OK cells [35], although the receptor numbers are similar in both cases (100-150fmol receptor/rag protein). This highlights, yet again, the critical role played by specialized G-protein subtypes and effector systems in signalling efficiency.

Expression of the majority of cloned 5HT receptors has been relatively straightforward. For example, mode> ate to high expression of 5HT1 receptors has been achieved in COS [22,26], CliO [21,22], L-cells [20,24], HeLa [25], 3T3 cells [33"], and HEK 293 [23,28,31] cells. Data from all these systems show that 5HT1 receptors are negatively coupled to adenylyl cyclase but, notably, the degree of cAMP inhibition varies significantly from host to host, perhaps reflecting inefficient coupling or an inappropriate G-protein repertoire in the host ceils. To date, at least 16 subtypes of the G-protein ix subunit alone have b e e n defined. Positive coupling to cyclase has been demonstrated for the newly cloned St'-B17 receptor stably expressed in HEK-293 cells; in contrast, no coupling has b e e n detected w h e n this receptor is transfected into COS ceils [28]. Likewise, phosphatidyl inositol (PI)-coupling has b e e n demonstrated for receptors belonging to the 5HT2 class [27].

Interleukin-2 receptor Multimeric receptors pose a particular challenge to the investigator. They can be refractory to cloning if more than one chain is required for ligand binding and highlevel expression can also be difficult. Nonetheless, the flexibility of mammalian cell expression, permits detailed examination of receptor function.

The demonstration of effector coupling has, however, proved to be a problem with 5HT5 receptors. This subfamily consists of the A and B subtypes [29,30]. Expression of the 5HT5A receptor in NIH-3T3 ceils resuits in two distinct populations of high- and low-affinity states, with the high-affinity sites showing 5HTID-

The interleukin (IL)-2 receptor plays a role in the proliferation and differentiation of T and B ceils, natural killer cells, and cells derived from the monocytic lineage [36,37]. As far back as 1989, it was k n o w n to exist in three affinity states (high, middle and low) and to b e generated by at least two (ix and ~) subunits [37].

Expressionof mammalian cell-surfacereceptors Browne and Balcarek Early attempts at mammalian cell expression of the two cloned cx and 13 chains, however, met with only limited success. It was found that co-expression of c~ and chains in fibroblast cells resulted in high-affinity binding sites incapable of a functional response, whereas the expression in lymphocytes of the ~ chain alone resulted in the formation of a functional site of intermediate affinity [38]. Such discrepancies inter alia suggested a third component was necessary for reconstitution of the functional high- and intermediate-affinity IL-2 receptor. Takeshita et al, [39"q have recently reported the cloning and expression of this third component, the 7 chain. In a series of co-transfection experiments they show: first, the high-affinity site is only obtained in a~y-transfectants; second, the intermediate-affinity site appears to be formed by the ~ and 3' subunits; and third, the low-affinity site is formed b y expression of ~ subunits. Moreover, the presence of the y chain is necessary for rapid internalization of IL-2 following binding to the receptor.

The baculovirus system While vertebrate-based expression systems are extremely versatile, the baculovirus system (see Luckow pp 564-572) has proved attractive as a means of producing large quantities of receptor (e.g. m2 muscarinic acetylcholine receptor at 20 to 30 pmole receptor/mg protein) relatively quickly, without the n e e d to create stable cell lines [40]. Baculovirus is gaining a reputation for being capable of handling proteins that are intractable to other systems. In part, this may be a result of the 'transient' nature of expression. Indeed, the baculovirus system has permitted expression of the 'problematical' hD4 receptor at levels of 5 pmole receptor/mg protein [15-']; interestingly, expression is only detectable when the cDNA is fused to the first 11 amino acids of the polyhedrin protein. Baculovirus also appears to allow functional responses to some receptors. Stimulation of endogenous adenylyl cyclase has been demonstrated u p o n expression of turkey or hamster ~2 adrenergic receptor [40], and the rat m3 muscarinic receptor is capable of coupling with endogenous G proteins to activate potassium channels [41], paralleling earlier work carried out in mammalian cells. Demonstrably, at least some of the insect G proteins are sufficiently homologous to those of vertebrates to allow efficient coupling to take place. Baculovirus has been similarly useful in expressing multimeric receptors to a high level. GABAA receptors are ligand-gated C1- channels of immense pharmacological interest and are believed (by analogy with nicotinic acetylcholine receptors) to be heteropentameric structures, which contain binding sites for benzodiazepines, barbiturates and alcohol. These compounds are believed to act as modulators of receptor function

[421. Cloning of cDNA has revealed a large family of receptor subunits (six ~ subunits, four ~ subunits, three 3' subunits, one 8 subunit and two p subunits, to date) capable of generating a wide degree of subtype diversity [42,43]. When 0tll~272 or 0t3~272 subunits are introduced into insect cells, expression levels of 3pmol receptor/mg protein (high-affinity sites) are achieved [44]. This compares with the relatively low levels (100-200 fmol receptor/mg protein) achieved in transient expression systems (HEK-293 ceils [45], oocytes [46]). Importantly, the infected ceils express GABAresponsive C1- channels showing benzodiazepine sensitivity, with a pharmacological profile similar to that observed in animal cells.

Novel systems for detecting signal transduction In most instances, effector coupling and signal transduction of transfected receptors are assayed by 'traditional' biochemical methods (e.g. adenylyl cyclase activity, cAMP modulation, and ion flux). While these methods are useful, they are often labour intensive. Three systems have recently b e e n described that use novel approaches to detect receptor-mediated signal transduction and which could have wide-spread utility. Sheu et al. [47"q have reported the use of the bioluminescent protein aequorin to measure 5HT-stimulated Ca 2+ release in HEK-293 ceils expressing the rat 5HT2 receptor. Aequorin is a complex consisting of apoaequorin, coelenterazine and bound oxygen, which emits light u p o n binding Ca 2+. Following co-transfection of the rat 5HT2 receptor and jellyfish apoaequorin, aequorin is reconstituted by incubating the cells with coelenterizine; the cells luminesce following 5-HT stimulation. Furthermore, the response is dose-dependent, saturable and inhibited by 5-HT antagonism. Cell lines stably expressing aequorin have b e e n made and should provide a convenient assay for measuring ligand-stimulated changes in intracellular Ca 2+ concentration. Exciting advances have also b e e n made using a clonal X e n o p u s laevis melanophore cell line [48]. A number of recombinant receptors (e.g. hD2, human ~i2 adrenergic receptor, murine substance P receptor and murine bombesin receptor) have b e e n expressed in these ceils. The receptors appear to couple appropriately to adenylyl cyclase and phospholipase C, and importantly, ligand binding results in changes in the movement of melanosomes (pigmented organelles), which can be monitored b y video imaging. In a similar approach, mouse Y1 cells microinjected with a [32 adrenergic receptor have b e e n shown to respond by major morphological changes to isoproterenol [49]. We await further development of these elegant and potentially powerful systems.

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Expressionsystems Conclusions N u m e r o u s receptor s y s t e m s operate in higher vertebrates, varying r e m a r k a b l y in structural complexity a n d function. While some r e c o m b i n a n t receptors a p p e a r to b e e x p r e s s e d and c o u p l e d with f u n c t i o n relatively easily, others are more problematical. Unfortunately, no certain m e a n s is currently available for predicting w h i c h route will s u c c e e d and it m a y b e necessary to try several systems to e n s u r e success. O f course, o n e always n e e d s to be mindful of the n e e d to relate studies in these m o d e l r e c o m b i n a n t systems to the native situation.

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F o r t h e f u t u r e , it is i m p o r t a n t to i m p r o v e u p o n c u r r e n t systems. We expect to see both continued studies on c o - e x p r e s s i o n o f G p r o t e i n s in a v a r i e t y o f cell t y p e s , w h i c h w i l l facilitate f u n c t i o n a l s t u d i e s o f n o v e l r e c e p t o r s [50], a n d f u r t h e r e x p l o r a t i o n o f t h e X e n o p u s a n d insect cell systems. We a l s o f o r e s e e f u r t h e r d e v e l o p m e n t of novel rapid and micro-scale methods for measuring functional responses of cell-surface receptors.

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MJ Browne, Department of Biotechnology, SmithKlme Beecham Pharmaceuticals, The Frythe, Welwyn, Hertfordshire, AL6 9AR, UK. JM Balcarek, Department of Molecular Genetics, SmitbKline Beecham Pharmaceuticals, Upper Menon, Pennsylvania 19406, USA.

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