Chimaeric Constructs Research Articles

Expression of genes encoding steroidogenic enzymes in the bovine corpus luteum

To examine the regulation of P-450SCC expression at the molecular level, a transfection protocol specific for bovine luteal cell cultures was developed. Among several commonly used transfection methods, electroporation yielded highest transfection efficiencies. Transfection of primary cultures of bovine luteal cells with chimaeric DNA constructs containing increasing deletions of the 5'-flanking region of P-450SCC fused to the chloramphenicol acetyl transferase (CAT) reporter gene allowed two conclusions. Firstly, sequences of the P-450SCC 5'-flanking region were capable of conferring basal expression and cAMP responsiveness to the CAT reporter gene and secondly, the region -186 to -100 bp appears to be required for these two types of regulation of gene expression. On the other hand, 5'-flanking regions of P-450(17 alpha) were not capable of conferring any regulation of gene expression to the CAT reporter gene in these cells. Thus, the physiologically observed regulation of the endogenous cytochromes P-450SCC and P-450(17 alpha) is closely reflected by CAT reporter gene expression. These experiments will allow investigation of the molecular mechanisms underlying the regulation of the genes encoding steroidogenic enzymes throughout the ovarian cycle.

Variations in CYP78A13 coding region influence grain size and yield in rice.

Grain size is one of the most important determinants of crop yield in cereals. Here, we identified a dominant mutant, big grain2 (bg2-D) from our enhancer-trapping population. Genetic analysis and SiteFinding PCR (polymerase chain reaction) revealed that BG2 encodes a cytochrome P450, OsCYP78A13. Sequence search revealed that CYP78A13 has a paralogue Grain Length 3.2 (GL3.2, LOC_Os03g30420) in rice with distinct expression patterns, analysis of transgenic plants harbouring either CYP78A13 or GL3.2 showed that both can promote grain growth. Sequence polymorphism analysis with 1529 rice varieties showed that the nucleotide diversity at CYP78A13 gene body and the 20 kb flanking region in the indica varieties were markedly higher than those in japonica varieties. Further, comparison of the genomic sequence of CYP78A13 in the japonica cultivar Nipponbare and the indica cultivar 9311 showed that there were three InDels in the promoter region and eight SNPs (single nucleotide polymorphism) in its coding sequence. Detailed examination of the transgenic plants with chimaeric constructs suggested that variation in CYP78A13 coding region is responsible for the variation of grain yield. Taken together, our results suggest that the variations in CYP78A13 in the indica varieties hold potential in rice breeding for application of grain yield improvement.

Signal-peptide-peptidase-like 2a (SPPL2a) is targeted to lysosomes/late endosomes by a tyrosine motif in its C-terminal tail

Signal-peptide-peptidase-like 2A (SPPL2a), an aspartyl intramembrane protease, has been implicated in the proteolysis of TNF-alpha, Fas Ligand and Bri2. Here, we show that endogenous SPPL2a – in agreement with overexpression studies – is localised in membranes of lysosomes/late endosomes. Furthermore, we have analysed the molecular determinants for lysosomal sorting of SPPL2a by creating chimaeric constructs between SPPL2a and its plasma membrane localised homologue SPPL2b. Lysosomal transport of SPPL2a critically depends on its cytosolic carboxyterminal tail. A canonical tyrosine-based sorting motif of the YXXø type at position 498 is sufficient to direct SPPL2a to lysosomal/late endosomal compartments. This motif accounts for the differential localisation of the homologous proteases SPPL2a and SPPL2b and thereby influences the access to substrates and biological function of SPPL2a. Structured summary of protein interactionsLAMP2 and SPPL2acolocalize by fluorescence microscopy(view interaction)

Different domains are critical for oligomerization compatibility of different connexins

Oligomerization of connexins is a critical step in gap junction channel formation. Some members of the connexin family can oligomerize with other members and form functional heteromeric hemichannels [e.g. Cx43 (connexin 43) and Cx45], but others are incompatible (e.g. Cx43 and Cx26). To find connexin domains important for oligomerization, we constructed chimaeras between Cx43 and Cx26 and studied their ability to oligomerize with wild-type Cx43, Cx45 or Cx26. HeLa cells co-expressing Cx43, Cx45 or Cx26 and individual chimaeric constructs were analysed for interactions between the chimaeras and the wild-type connexins using cell biological (subcellular localization by immunofluorescence), functional (intercellular diffusion of microinjected Lucifer yellow) and biochemical (sedimentation velocity through sucrose gradients) assays. All of the chimaeras containing the third transmembrane domain of Cx43 interacted with wild-type Cx43 on the basis of co-localization, dominant-negative inhibition of intercellular communication, and altered sedimentation velocity. The same chimaeras also interacted with co-expressed Cx45. In contrast, immunofluorescence and intracellular diffusion of tracer suggested that other domains influenced oligomerization compatibility when chimaeras were co-expressed with Cx26. Taken together, these results suggest that amino acids in the third transmembrane domain are critical for oligomerization with Cx43 and Cx45. However, motifs in different domains may determine oligomerization compatibility in members of different connexin subfamilies.

Two dileucine motifs mediate late endosomal/lysosomal targeting of transmembrane protein 192 (TMEM192) and a C-terminal cysteine residue is responsible for disulfide bond formation in TMEM192 homodimers

TMEM192 (transmembrane protein 192) is a novel constituent of late endosomal/lysosomal membranes with four potential transmembrane segments and an unknown function that was initially discovered by organellar proteomics. Subsequently, localization in late endosomes/lysosomes has been confirmed for overexpressed and endogenous TMEM192, and homodimers of TMEM192 linked by disulfide bonds have been reported. In the present study the molecular determinants of TMEM192 mediating its transport to late endosomes/lysosomes were analysed by using CD4 chimaeric constructs and mutagenesis of potential targeting motifs in TMEM192. Two directly adjacent N-terminally located dileucine motifs of the DXXLL-type were found to be critical for transport of TMEM192 to late endosomes/lysosomes. Whereas disruption of both dileucine motifs resulted in mistargeting of TMEM192 to the plasma membrane, each of the two motifs was sufficient to ensure correct targeting of TMEM192. In order to study disulfide bond formation, mutagenesis of cysteine residues was performed. Mutation of Cys266 abolished disulfide bridge formation between TMEM192 molecules, indicating that TMEM192 dimers are linked by a disulfide bridge between their C-terminal tails. According to the predicted topology, Cys266 would be localized in the reductive milieu of the cytosol where disulfide bridges are generally uncommon. Using immunogold labelling and proteinase protection assays, the localization of the N- and C-termini of TMEM192 on the cytosolic side of the late endosomal/lysosomal membrane was experimentally confirmed. These findings may imply close proximity of the C-termini in TMEM192 dimers and a possible involvement of this part of the protein in dimer assembly.

N-terminal chimaeras with signal sequences enhance the functional expression and alter the subcellular localization of heterologous membrane-bound inorganic pyrophosphatases in yeast

Expression of heterologous multispanning membrane proteins in Saccharomyces cerevisiae is a difficult task. Quite often, the use of multicopy plasmids where the foreign gene is under the control of a strong promoter does not guarantee efficient production of the corresponding protein. In the present study, we show that the expression level and/or subcellular localization in S. cerevisiae of a heterologous type of multispanning membrane protein, the proton-translocating inorganic pyrophosphatase (H+-PPase), can be changed by fusing it with various suitable N-terminal signal sequences. Chimaeric proteins were constructed by adding the putative N-terminal extra domain of Trypanosoma cruzi H+-PPase or the bona fide signal sequence of S. cerevisiae invertase Suc2p to H+-PPase polypeptides of different organisms (from bacteria to plants) and expressed in a yeast conditional mutant deficient in its cytosolic PPi hydrolysis activity when grown on glucose. Chimaeric constructs not only substantially enhanced H+-PPase expression levels in transformed mutant cells, but also allowed functional complementation in those cases in which native H+-PPase failed to accomplish it. Activity assays and Western blot analyses demonstrated further the occurrence of most H+-PPase in internal membrane fractions of these cells. The addition of N-terminal signal sequences to the vacuolar H+-PPase AVP1 from the plant Arabidopsis thaliana, a protein efficiently expressed in yeast in its natural form, alters the subcellular distribution of the chimaeras, suggesting further progression along the secretory sorting pathways, as shown by density gradient ultracentrifugation and in vivo fluorescence microscopy of the corresponding GFP (green fluorescent protein)-H+-PPase fusion proteins.

Two interaction sites on mammalian adenylyl cyclase type I and II: modulation by calmodulin and Gβγ

Mammalian ACs (adenylyl cyclases) are integrating effector molecules in signal transduction regulated by a plethora of molecules in either an additive, synergistic or antagonistic manner. Out of nine different isoforms, each AC subtype uses an individual set of regulators. In the present study, we have used chimaeric constructs, point mutations and peptide competition studies with ACs to show a common mechanism of multiple contact sites for the regulatory molecules G(betagamma) and calmodulin. Despite their chemical, structural and functional variety and different target motifs on AC, G(betagamma) and calmodulin share a two-site-interaction mechanism with G(alphas) and forskolin to modulate AC activity. Forskolin and G(alphas) are known to interact with both cytosolic domains of AC, from inside the catalytic cleft as well as at the periphery. An individual interaction site located at C(1) of the specifically regulated AC subtype had been ascribed for both G(betagamma) and calmodulin. In the present study we now show for these two regulators of AC that a second isoform- and regulator-specific contact site in C(2) is necessary to render enzyme activity susceptible to G(betagamma) or calmodulin modulation. In addition to the PFAHL motif in C(1b) of ACII, G(betagamma) contacts the KF loop in C(2), whereas calmodulin requires not only the Ca2+-independent AC28 region in C(1b) but also a Ca2+-dependent domain in C(2a) of ACI containing the VLG loop to stimulate this AC isoform.

Identification of a region on hypoxia-inducible-factor prolyl 4-hydroxylases that determines their specificity for the oxygen degradation domains

HIFs [hypoxia-inducible (transcription) factors] are essential for the induction of an adaptive gene expression programme under low oxygen partial pressure. The activity of these transcription factors is mainly determined by the stability of the HIFalpha subunit, which is regulated, in an oxygen-dependent manner, by a family of three prolyl 4-hydroxylases [EGLN1-EGLN3 (EGL nine homologues 1-3)]. HIFalpha contains two, N- and C-terminal, independent ODDs (oxygen-dependent degradation domains), namely NODD and CODD, that, upon hydroxylation by the EGLNs, target HIFalpha for proteasomal degradation. In vitro studies indicate that each EGLN shows a differential preference for ODDs, However, the sequence determinants for such specificity are unknown. In the present study we showed that whereas EGLN1 and EGLN2 acted upon any of these ODDs to regulate HIF1alpha protein levels and activity in vivo, EGLN3 only acted on the CODD. With the aim of identifying the region within EGLNs responsible for their differential substrate preference, we investigated the activity and binding pattern of different EGLN deletions and chimaeric constructs generated by domain swapping between EGLN1 and EGLN3. These studies revealed a region of 97 residues that was sufficient to confer the characteristic substrate binding observed for each EGLN. Within this region, we identified the minimal sequence (EGLN1 residues 236-252) involved in substrate discrimination. Importantly, mapping of these sequences on the EGLN1 tertiary structure indicates that substrate specificity is determined by a region relatively remote from the catalytic site.

A Di‐Acidic Sequence Motif Enhances the Surface Expression of the Potassium Channel TASK‐3

We have characterized a sequence motif, EDE, in the proximal C-terminus of the acid-sensitive potassium channel TASK-3. Human TASK-3 channels were expressed in Xenopus oocytes, and the density of the channels at the surface membrane was studied with two complementary techniques: a luminometric surface expression assay of hemagglutinin epitope-tagged TASK-3 channels and voltage-clamp measurements of the acid-sensitive potassium current. Both approaches showed that mutation of the two glutamate residues of the EDE motif to alanine (ADA mutant) markedly reduced the transport of TASK-3 channels to the cell surface. Mutation of the central aspartate of the EDE motif had no effect on surface expression. The functional role of the EDE motif was further characterized in chimaeric constructs consisting of truncated Kir2.1 channels to which the C-terminus of TASK-3 was attached. In these constructs, too, replacement of the EDE motif by ADA strongly reduced surface expression. Live-cell imaging of enhanced green fluorescent protein-tagged channels expressed in COS-7 cells showed that 24 h after transfection wild-type TASK-3 was mainly localized to the cell surface whereas the ADA mutant was largely retained in the endoplasmic reticulum (ER). Mutation of a second di-acidic motif in the C-terminus of TASK-3 (DAE) had no effect on surface expression. Coexpression of TASK-3 with a GTP-restricted mutant of the coat recruitment GTPase Sar1 (Sar1H79G) resulted in ER retention of the channel. Our data suggest that the di-acidic motif, EDE, in human TASK-3 is a major determinant of the rate of ER export and is required for efficient surface expression of the channel.

Dynamics of bidirectional transport of Arc mRNA in neuronal dendrites

The mRNA for Arc (activity-regulated cytoskeletal protein) is delivered into dendrites and localizes selectively at active synapses. Here we use a green fluorescent protein-based labeling system and confocal microscopy to define the transport kinetics of exogenously expressed mRNA from chimaeric Arc constructs (Arc/MS2 mRNA) in the dendrites of living rat neurons in culture. Arc/MS2 mRNA assembles into particles that move independently, bidirectionally, and intermittently in a fashion indicative of transport. Transport velocities range from below 6 to 65 mum/minute, which is consistent with actin-based and microtubule-based transport, respectively. In general, orthograde translocations are longer than retrograde translocations. Rapidly translocating Arc/MS2 mRNA particles sometimes reverse direction and decrease velocity just before stopping, suggesting that local signals regulate Arc mRNA targeting movements. These observations identify several phases of Arc mRNA movement that serve as potential points for regulating Arc mRNA localization.

Novel structural determinants of single-channel conductance in nicotinic acetylcholine and 5-hydroxytryptamine type-3 receptors

Nicotinic ACh (acetylcholine) and 5-HT3 (5-hydroxytryptamine type-3) receptors are cation-selective ion channels of the Cys-loop transmitter-gated ion channel superfamily. Numerous lines of evidence indicate that the channel lining domain of such receptors is formed by the alpha-helical M2 domain (second transmembrane domain) contributed by each of five subunits present within the receptor complex. Specific amino acid residues within the M2 domain have accordingly been demonstrated to influence both single-channel conductance (gamma) and ion selectivity. However, it is now clear from work performed on the homomeric 5-HT3A receptor, heteromeric 5-HT3A/5-HT3B receptor and 5-HT3A/5-HT3B receptor subunit chimaeric constructs that an additional major determinant of gamma resides within a cytoplasmic domain of the receptor termed the MA-stretch (membrane-associated stretch). The MA-stretch, within the M3-M4 loop, is not traditionally thought to be implicated in ion permeation and selection. Here, we describe how such observations extend to a representative neuronal nicotinic ACh receptor composed of alpha4 and beta2 subunits and, by inference, probably other members of the Cys-loop family. In addition, we will attempt to interpret our results within the context of a recently developed atomic scale model of the nicotinic ACh receptor of Torpedo marmorata (marbled electric ray).

Archaeal Pyrococcus furiosus thymidylate synthase 1 is an RNA-binding protein

Using a stem-loop RNA oligonucleotide (19-mer) containing an AUG sequence in the loop region as a probe, we screened the protein library from a hyperthermophilic archaeon, Pyrococcus furiosus, and found that a flavin-dependent thymidylate synthase, Pf-Thy1 (Pyrococcus furiosus thymidylate synthase 1), possessed RNA-binding activity. Recombinant Pf-Thy1 was able to bind to the stem-loop structure at a high temperature (75 degrees C) with an apparent dissociation constant of 0.6 microM. A similar stem-loop RNA structure was located around the translation start AUG codon of Pf-Thy1 RNA, and gel-shift analysis revealed that Pf-Thy1 could also bind to this stem-loop structure. In vitro translation analysis using chimaeric constructs containing the stem-loop sequence in their Pf-Thy1 RNA and a luciferase reporter gene indicated that the stem-loop structure acted as an inhibitory regulator of translation by preventing the binding of its Shine-Dalgarno-like sequence by positioning it in the stem region. Addition of Pf-Thy1 into the in vitro translation system also inhibited translation. These results suggested that this class of thymidylate synthases may autoregulate their own translation in a manner analogous to that of the well characterized thymidylate synthase A proteins, although there is no significant amino acid sequence similarity between them.

The requirement of yeast replication origins for pre-replication complex proteins is modulated by transcription

The mini-chromosome maintenance proteins Mcm2–7 are essential for DNA replication. They are loaded onto replication origins during G1 phase of the cell cycle to form a pre-replication complex (pre-RC) that licenses each origin for subsequent initiation. We have investigated the DNA elements that determine the dependence of yeast replication origins on Mcm2–7 activity, i.e. the sensitivity of an origin to mcm mutations. Using chimaeric constructs from mcm sensitive and mcm insensitive origins, we have identified two main elements affecting the requirement for Mcm2–7 function. First, transcription into an origin increases its dependence on Mcm2–7 function, revealing a conflict between pre-RC assembly and transcription. Second, sequence elements within the minimal origin influence its mcm sensitivity. Replication origins show similar differences in sensitivity to mutations in other pre-RC proteins (such as Origin Recognition Complex and Cdc6), but not to mutations in initiation and elongation factors, demonstrating that the mcm sensitivity of an origin is determined by its ability to establish a pre-RC. We propose that there is a hierarchy of replication origins with respect to the range of pre-RC protein concentrations under which they will function. This hierarchy is both ‘hard-wired’ by the minimal origin sequences and ‘soft-wired’ by local transcriptional context.

Optimization of factor VIII replacement therapy: can structural studies help in evading antibody inhibitors?

Optimization of factor VIII replacement therapy: can structural studies help in evading antibody inhibitors?

Chimaeras reveal the role of the catalytic core in the activation of the plasma membrane Ca2+ pump

Isoform 2b of the plasma membrane calcium pump differs from the ubiquitous isoform 4b in the following: (a) higher basal activity in the absence of calmodulin; (b) higher affinity for calmodulin; and (c) higher affinity for Ca2+ in the presence of calmodulin [Elwess, Filoteo, Enyedi and Penniston (1997) J. Biol. Chem. 272, 17981–17986]. To investigate which parts of the molecule determine these kinetic differences, we made four chimaeric constructs in which portions of isoform 2b were grafted into isoform 4b: chimaera I contains only the C-terminal regulatory region of isoform 2b; chimaera II contains the N-terminal moiety of isoform 2b, including both cytoplasmic loops; chimaera III contains the sequence of isoform 2b starting from the N-terminus to after the end of the first (small) cytoplasmic loop; and chimaera IV contains only the second (large) cytoplasmic loop. Surprisingly, chimaera I showed low basal activity in the absence of calmodulin and low affinity for calmodulin, unlike isoform 2b. In contrast, the chimaera containing both loops showed high basal activity, and Ca2+ activation curves (both in the absence and in the presence of calmodulin) similar to those of isoform 2b. The rates of activation by calmodulin and of inactivation by calmodulin removal were measured, and the apparent Kd for calmodulin was calculated from the ratio between these rate constants. The order of affinity was: 2b = II>4b = IV>III = I. From these results it is clear that the construct that most closely resembles isoform 2b is chimaera II. This shows that, in order to obtain an enzyme with properties similar to those of isoform 2b, both cytoplasmic loops are needed.

Chimaeras reveal the role of the catalytic core in the activation of the plasma membrane Ca2+ pump.

Isoform 2b of the plasma membrane calcium pump differs from the ubiquitous isoform 4b in the following: (a) higher basal activity in the absence of calmodulin; (b) higher affinity for calmodulin; and (c) higher affinity for Ca(2+) in the presence of calmodulin [Elwess, Filoteo, Enyedi and Penniston (1997) J. Biol. Chem. 272, 17981-17986]. To investigate which parts of the molecule determine these kinetic differences, we made four chimaeric constructs in which portions of isoform 2b were grafted into isoform 4b: chimaera I contains only the C-terminal regulatory region of isoform 2b; chimaera II contains the N-terminal moiety of isoform 2b, including both cytoplasmic loops; chimaera III contains the sequence of isoform 2b starting from the N-terminus to after the end of the first (small) cytoplasmic loop; and chimaera IV contains only the second (large) cytoplasmic loop. Surprisingly, chimaera I showed low basal activity in the absence of calmodulin and low affinity for calmodulin, unlike isoform 2b. In contrast, the chimaera containing both loops showed high basal activity, and Ca(2+) activation curves (both in the absence and in the presence of calmodulin) similar to those of isoform 2b. The rates of activation by calmodulin and of inactivation by calmodulin removal were measured, and the apparent K(d) for calmodulin was calculated from the ratio between these rate constants. The order of affinity was: 2b=II>4b=IV>III=I. From these results it is clear that the construct that most closely resembles isoform 2b is chimaera II. This shows that, in order to obtain an enzyme with properties similar to those of isoform 2b, both cytoplasmic loops are needed.

Identification of an extracellular segment of the oxytocin receptor providing agonist-specific binding epitopes

The effects of the peptide hormone oxytocin are mediated by oxytocin receptors (OTRs) expressed by the target tissue. The OTR is a member of the large family of G-protein-coupled receptors. Defining differences between the interaction of agonists and antagonists with the OTR at the molecular level is of fundamental importance, and is addressed in this study. Using truncated and chimaeric receptor constructs, we establish that a small 12-residue segment in the distal portion of the N-terminus of the human OTR provides important epitopes which are required for agonist binding. In contrast, this segment does not contribute to the binding site for antagonists, whether peptide or non-peptide. It does, however, have a role in agonist-induced OTR signalling. Oxytocin is also an agonist at the vasopressin V1a receptor (V1aR). A chimaeric receptor (V1aRN-OTR) was engineered in which the N-terminus of the OTR was substituted by the corresponding, but unrelated, sequence from the N-terminus of the V1aR. We show that the V1aR N-terminus present in V1aRN-OTR fully restored both agonist binding and intracellular signalling to a dysfunctional truncated OTR construct. The N-terminal segment does not, however, contribute to receptor-selective agonism between the OTR and the V1aR. Our data establish a key role for the distal N-terminus of the OTR in providing agonist-specific binding epitopes.

Analysis of uracil-DNA glycosylases from the murine Ung gene reveals differential expression in tissues and in embryonic development and a subcellular sorting pattern that differs from the human homologues.

The murine UNG: gene encodes both mitochondrial (Ung1) and nuclear (Ung2) forms of uracil-DNA glyco-sylase. The gene contains seven exons organised like the human counterpart. While the putative Ung1 promoter (P(B)) and the human P(B) contain essentially the same, although differently organised, transcription factor binding elements, the Ung2 promoter (P(A)) shows limited homology to the human counterpart. Transient transfection of chimaeric promoter-luciferase constructs demonstrated that both promoters are functional and that P(B) drives transcription more efficiently than P(A). mRNAs for Ung1 and Ung2 are found in all adult tissues analysed, but they are differentially expressed. Furthermore, transcription of both mRNA forms, particularly Ung2, is induced in mid-gestation embryos. Except for a strong conservation of the 26 N-terminal residues in Ung2, the subcellular targeting sequences in the encoded proteins have limited homology. Ung2 is transported exclusively to the nucleus in NIH 3T3 cells as expected. In contrast, Ung1 was sorted both to nuclei and mitochondria. These results demonstrate that although the catalytic domain of uracil-DNA glycosylase is highly conserved in mouse and man, regulatory elements in the gene and subcellular sorting sequences in the proteins differ both structurally and functionally, resulting in altered contribution of the isoforms to total uracil-DNA glycosylase activity.

The α-isoform of class II phosphoinositide 3-kinase is more effectively activated by insulin receptors than IGF receptors, and activation requires receptor NPEY motifs

Little is known about the physiological role and mechanism of activation of class II phosphoinositide 3-kinases (PI3Ks), although it has been shown that the PI3K-C2α isoform is activated by insulin. Using chimaeric receptor constructs which can be activated independently of endogenous receptors in transfected cells, we found that PI3K-C2α activity was stimulated to a greater extent by insulin receptors than IGF receptors in 3T3-L1 adipocytes. Activation of PI3K-C2α required an intact NPEY motif in the receptor juxtamembrane domain. We conclude that PI3K-C2α is a candidate for participation in insulin-specific intracellular signalling.

Domains determining agonist selectivity in chimaeric VIP2 (VPAC2)/PACAP (PAC1) receptors.

1 The VPAC2 and PAC1 receptors are closely related members of the Group II G protein-coupled receptor family. At the VPAC2 receptor, VIP is equipotent to PACAP-38 in stimulating cyclic AMP production, whereas at the PAC1 receptor PACAP-38 is many fold more potent than VIP. In this study, domains which confer this selectivity were investigated by constructing four chimaeric receptors in which segments of the VPAC2 receptor were exchanged with the corresponding segment from the PAC1 receptor. 2 When expressed in COS 7 cells all the chimaeric receptors bound the common ligand [125I]PACAP-27 and produced cyclic AMP in response to agonists. 3 Relative selectivity for agonists was determined primarily by the amino terminal extracellular domain of the PAC1 receptor and the VPAC2 receptor. The interchange of other domains had little effect on the potency of PACAP-38 or PACAP-27. 4 For chimaeric constructs with a PAC1 receptor amino terminal domain, the substitution of increasing portions of the VPAC2 receptor decreased the potency of VIP yet increased that of helodermin. 5 This suggests that the interaction of VIP/helodermin but not PACAP with the PAC1 receptor may be influenced (and differentially so) by additional receptor domains.