The long and the short of reporter gene assays

A typical series of experiments aimed at understanding the basis of a particular GPCR expression pattern might go like this: (1) isolate a genomic fragment containing the GPCR gene,

(2) make a series of reporter gene constructs, transiently transfect these into expressing and non-expressing cell lines and measure reporter gene activity to localize regulatory domains,

(3) identify individual regulatory elements using protein/DNA interaction assays such as electromobility shift assays and DNase footprinting, and (4) use these elements to identify cognate transcription factors using gel shifts, screening expression libraries or one-hybrid assays. So what is wrong with that? There are several potential pitfalls to this approach—but they should not obscure its general usefulness. First, templates are present in transiently transfected cells as high copy episomes of many thousands (although only a small fraction are transcriptionally competent), whereas only two copies of an endogenous GPCR gene are found in a diploid cell (Smith and Hager 1997). Second, chromatin organization on episomal DNA differs from that of chromosomal genes (Archer et al. 1992; Smith et al. 1997). Third, most cell lines offer only a poor representation of their natural untransformed counterparts and this may be reflected in the molecules and mechanisms recruited to control transcription. Nevertheless, such experiments do yield valuable data and the bulk of the information that we have on regulation of GPCR transcription stems from these types of studies. Rather than seeing such experiments as definitive it is better to see them as enabling—providers of candidate regulatory elements whose role needs to be verified in a more physiological setting.

The promoters of a number of GPCRs have been examined over the last decade. There is neither space to attempt to comprehensively assess each of these studies nor justification to present such a catalogue. Instead, we have tabulated a selection of key references according to receptor type (Table 2.1). The focus presented here is upon selected examples that serve to illustrate general principles or even cautionary tales. Particular attention has been given to studies that illustrate the role of transcription in regulating endogenous GPCR expression. Many of these points can be illustrated by work carried out over the last six years by ourselves and others on the regulation of the M4 muscarinic receptor gene.

Table 2.1 Transcriptional regulation of GPCR genes: Examples have been chosen where interaction of a known transcription factor has been demonstrated by interaction either on the basis of a functional assay (using either the endogenous gene or a reporter gene) or biochemical interaction (such as electromobility shift assays, chromatin immunoprecipitation, or DNase footprinting). Studies that have demonstrated only relevant cis regulatory regions of a gene have not been included

Table 2.1 Transcriptional regulation of GPCR genes: Examples have been chosen where interaction of a known transcription factor has been demonstrated by interaction either on the basis of a functional assay (using either the endogenous gene or a reporter gene) or biochemical interaction (such as electromobility shift assays, chromatin immunoprecipitation, or DNase footprinting). Studies that have demonstrated only relevant cis regulatory regions of a gene have not been included

Receptor

Proposed regulatory elements/transcription factors

D1A dopamine

AP-2 (Takeuchi et al. 1993) Sp1; Zic2 (Yang et al. 2000)

D2 dopamine

AP-1 (Valdenaire etal. 1994; Wang etal. 1997)

B1 bradykinin

NFkB/CRE (Yang and Polgar 1996) AP-1 (Yang etal. 1998b)

B2 bradykinin

p53; p300/CBP (Saifudeen etal. 2000)

P2 adrenergic

GRE/GR (Malbon and Hadcock 1988)

adrenergic

RARE/TH (Bahouth et al. 1997) GR (Tseng and Padbury 2000)

p, opioid

PPY tract (Ko and Loh 2001)

A1 adenosine

GATA; Nkx2.5 (Rivkees etal. 1999) GR (Ren and Stiles 1999)

VPAC1

(Karacay et al. 2000)

sst2

Smad4 (Puente etal. 2001)

PPT-1

CRE (Qian etal. 2001)

GRP-R

CRE (Weber et al. 2000)

M1 muscarinic

PPY (Wood et al. 1999)

M2 muscarinic

GATA; LIF/CNTF (Rosoff and Nathanson 1998; Rosoff etal. 1996)

M4 muscarinic

NRSF (Wood etal. 1996)

NPY1

NFkB (Musso etal. 1997)

Thrombin

Sp1 (Wu etal. 1998)

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