Yeast two-hybrid screening for proteins that interact with α₁-adrenergic receptors¹

Tan ZHANG, Qi XU, Feng-rong CHEN², Qi-de HAN, You-yi ZHANG³

Institute of Vascular Medicine, Peking University Third Hospital and Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Beijing 100083, China

¹ Project supported by the National Natural Science Foundation of China (30270540) and the Major State Basic Research Development Program of China (G2000056906).

² Now in Department of Cardiology, Peking University Third Hospital, Beijing, China.

³ Correspondence to Prof You-yi ZHANG. Phn 86-10-8280-2306. Fax 86-10-8280-2306. E-mail zhangyy@bjmu.edu.cn

Received 2004-01-12 Accepted 2004-05-20

KEY WORDS active Bcr-related protein; α₁-adrenergic receptors; bone morphogenetic protein-1; filamin-C; yeasts; two-hybrid system techniques; beta-galactosidase

ABSTRACT

AIM: To find novel proteins that may bind to α_1A-adrenergic receptor (α_1A-AR) and investigate their interactions with the other two α₁-AR subtypes (α_1B-AR and α_1D-AR) with an expectation to provide new leads for the function study of the receptors. METHODS: Yeast two-hybrid assay was performed to screen a human brain cDNA library using the C terminus of α_1A-AR (α_1A-AR-CT) as bait. X-Gal assay and o-nitrophenyl-beta-D-galactopyranoside (ONPG) assay were subsequently conducted to further qualitatively or quantitatively confirm the interactions between receptors and the three identified proteins. RESULTS: (1) Selection medium screening identified segments of bone morphogenetic protein-1 (BMP-1), active Bcr-related protein (Abr), and filamin-C as binding partners of α_1A-AR-CT in yeast cells respectively. Besides, protein segments of BMP-1 and Abr could only specifically interact with α_1A-AR-CT while filamin-C segment interacted with all three α₁-AR subtypes. (2) In X-Gal assay, the co-transformants of α_1A-AR-CT and BMP-1 segments turned strong blue at about 30 min while other positive transformants only developed weak blue at about 5-6 h. (3) In ONPG assay, interaction (shown in β-galactosidase activity) between α_1A-AR-CT and BMP-1 segments was about 30 times stronger than that of control (P<0.01), while other positive interactions were only about 2-5 times as strong as those of controls (P<0.05). CONCLUSION: In yeast cells BMP-1, Abr and/or filamin-C could interact with three α₁-AR subtypes, among which, interaction between BMP-1 and α_1A-AR was the strongest while other interactions between proteins and receptors were relatively weak.

INTRODUCTION

α₁-Adrenergic receptors (α₁-ARs) were members of G-protein-coupled receptor (GPCR) superfamily and consisted of α_1A-AR, α_1B-AR, and α_1D-AR subtypes. α₁-AR subtypes were important mediators of several cardiovascular actions regulated by the sympathetic nervous systems such as vasoconstriction, hypertrophy,cardiac inotropy, and remodeling^[1]. α₁-ARs were encoded by separate genes and they were different in structure, G protein coupling, tissue distribution, signaling, regulation, and functions. Among the three subtypes, it was clear that there was a high sequence homology (about 70 %) within the putative transmem brane domain. On the contrary, there was almost no sequence homology in the cytoplasmic C-terminal region. Recently, a variety of proteins have been found to regulate physiological functions of several adrenergic receptors by directly binding to their C-terminal regions. For example, gc1q-R, a multifunctional protein, was found to bind with the C-terminal cytoplasmic domain of the α_1B-AR^[2] and b₁-AR was found to be associated with the PSD-95, which facilitated the formation of a complex between b₁-AR and the N-methyl-D-aspartate receptors^[3]. Thus, from structural difference, we might explain functional differences existed among all the α₁-AR subtypes.

Considering the striking observations described above, we supposed that there might be a high possibility that some latent cytoplasmic proteins interacted with other ARs, including the α₁-AR. In all the three α₁-AR subtypes, it was reported that the vascular α_1A-AR played a role in the regulation of peripheral resistance and systemic arterial pressure, while α_1B-AR and α_1D-AR played minor roles in acute arterial pressure regulation. Besides, it was noted that α_1A-AR was the most efficient as to stimulate calcium release and inositol phosphate production whereas α_1D-AR was poorly coupled to intracellular signaling pathways ^[4]. So α_1A-AR played an important role in the regulation of cardiovascular functions. In this experiment, we screened a human brain cDNA library by yeast two-hybrid assay using α_1A-AR C-terminal domain as a bait, with an expectation to find new binding partners of α_1A-AR and thus provide clues for the functional study of α_1A-AR as well as the related proteins.

MATERIALS AND METHODS

Reagents Advantage 2 PCR kit, pGBKT7, Matchmaker 3 pretransformed human brain cDNA library, X-a-Gal and all other yeast two-hybrid components were purchased from Clontech. ECoR I, BamH I, T4 DNA ligase and ampicillin were purchased from Promega. PCR Product Purification Kit was obtained from Qiagen.

Plasmids construction The C-terminal domain of human α_1A-AR (amino acids 322-466), α_1B-AR (amino acids 344-519), and α_1D-AR (amino acids 398-572) was amplified respectively by polymerase chain reaction (PCR) from the full length human α_1A-AR, α_1B-AR, and α_1D-AR accordingly, using primer sets containing EcoR and BamH restriction sites (Tab 1), and was inserted downstream of the Gal4 DNA-binding domain in the (bait) vector pGBKT7 (CLONTECH Laboratories) using T4 DNA ligase respectively. All the three constructs (α_1A-AR-CT/pGBKT7, α_1B-AR-CT/pGBKT7, and α_1D-AR-CT/pGBKT7) were sequenced and the inserts were found to be in frame with the yeast Gal4 DNA-binding domain.

Tab 1. PCR primers used in this work.

a1A-AR-CT 5´F	5´-CCG GAATTC AAC CCC ATC ATA TAC CCA TGC TCC A-3´
a1A-AR-CT 3´R	5´-CGC GGATCC CCT GTC CTA GAC TTC CTC CCC GTT C-3´
a1B-AR-CT 5´F	5´-CG GAATTC AGG GGC ATG TTG GTT TTG AAG-3´
a1B-AR-CT 3´R	5´-CG GGATCC AAC CCC ATC ATC CCA TGC TC-3´
a1D-AR-CT 5´F	5´-CG GAATTC AAC CCG CTC ATC TAC CCC TGT TC-3´
a1D-AR-CT 3´R	5´-CG GGATCC AGG GAC ACA GCC TCC ACC TCT GA-3´

The underlined bases indicate restriction sites.

Yeast two-hybrid screening All assays were carried out according to the protocols described in the Pre-transformed MATCHMAKER Libraries User Manual (PT3183-1). In brief, the α_1A-AR-CT/pGBKT7 plasmids were initially introduced into the AH109 yeast strains using a modified lithium acetate protocol and the transformants were selected on SD/-Trp plates. The mating between the selected AH109 and Y187 yeast cells were performed and co-transformants were selected on agar plates SD/-Leu-Trp-His and SD/-Leu-His-Ade-Trp (QDO) with or without X-a-Gal to detect the initiation of reporter gene (HIS, LEU, TRP, ADE, and MEL1) transcription. Colonies grew at 30 ºC for about 5 d and those turned blue were at last selected as positive clones. Plasmids from positive clones were extracted, introduced parts of the cDNA were amplified by PCR using sequencing primers (CLONTECH Libraries), then PCR products were sequenced and blasted on Genebank.

X-Gal assay and ONPG assay In order to confirm the positive reactions both of the assays were performed in the Y187 yeast strains to detect the initiation of LacZ reporter gene transcription qualitatively and quantitatively as described before^[5,6].

In X-Gal assay, Colony-lift Filter assay was used to check the activity of β-galactosidase. Briefly, fresh colonies grown to about 1-3 mm in diameter were transferred completely to a sterile filter and submerged in a pool of liquid nitrogen for 10 s and thawed at room temperature, then it was put on a pre-soaked filter in the Z buffer/X-Gal solution (100 mL Z buffer, 0.27 mL b-mercaptoethanol, 1.67 mL X-gal stock solution; Z buffer: Nα₂HPO₄·7H₂O 16.1 g/L, NaH₂PO₄·H₂O 5.50 g/L, KCl 0.75 g/L, MgSO₄·H₂O 0.246 g/L, pH=7.0), then the filters were incubated at 30 ºC and the colors of colonies were checked periodically.

In ONPG assay, ONPG was used as the substrate of β-galactosidase for the liquid culture assay. In brief, at least three independent clones were selected, grown, harvested, centrifuged, and resuspended in Z buffer, frozen in liquid nitrogen, and thawed at 37 ºC in a water bath. Then the reaction systems (ONPG+Z buffer+b-mercaptoethanol+yeast cells resuspension) were placed in a 30 ºC incubator. After the yellow color developed Nα₂CO₃1 mol/L was added to the reaction and blank tubes. Relapsed time was recorded in minutes. Reaction tubes were centrifuged at 14 000 r/min for 10 min and supernatants were carefully transferred to clean cuvettes and OD₄₂₀ of the samples relative to the blank was recorded. At last, the β-galactosidase units were calculated as:

β-galactosidase units=1000×OD₄₂₀/(t× V×OD₆₀₀)

where t =elapsed time (in min) of incubation, V=0.1 mL×concentration factor (the concentration factor is 5), OD₆₀₀=Opital density at 600 nm of 1 mL of culture.

Statistical analysis All data were presented as mean±SD and processed with the SPSS v 11.0 statistical software package. Dunnett t-test was used for two-sided statistical analysis. The level of statistical significance was set at P<0.05.

RESULTS

Screening the binding partner of α_1A-AR C terminal domain in human brain cDNA library To identify novel proteins that interact with the α_1A-AR C terminal domain in vivo, we screened a human brain cDNA library subcloned into the bait vector pACT2 with the C-terminal domain of the α_1A-AR. Initially, to ensure that the α_1A-AR-CT/pGBKT7 clone alone was not able to activate the yeast reporter genes, the bait construct was co-transformed into the yeast AH109 strains with the library prey vector (pACT2) alone and tested for the transcription initiation of HIS and MEL1 reporter gene. The resulted clone was negative for self-activation and therefore, it was used as negative control in library screening.

Mating of the yeast Y187 strains was pre-transformed with a human brain library cDNA and the AH109 yeast strains containing α_1A-AR-CT/pGBKT7 plasmids was performed and approximately 2×10⁶ colonies among all the 2.3×10⁷ human brain cDNA library colonies were screened for α_1A-AR C-terminus. The library screening produced seven individual clones that were HIS⁺ and MEL1⁺ and could grow on the SD-Leu-His-Thr-Trp (QDO) agar plates and then they were subjected to sequence analysis. The blasting results showed that three of them encoded the C-terminal part of BMP-1 (Gene bank, GI: 5902809), Abr (Gene bank, GI: 15316553), and filamin-C (Gene bank, GI: 14744091) respectively. Co-transformation of α_1A-AR-CT/ pGBKT7 with pACT2 alone was also performed at the same time and the co-transformants could not grow on QDO selection medium, so the co-transformants of α_1A-AR-CT/pGBKT7 with pACT2 alone (1A-T) was used as control in this work. To eliminate false positive reactions, we co-transformed pGBKT7 with BMP-1/pACT2, Abr/pACT2, and filamin-C/pACT2 respectively, no transcription initiation of reporter gene was detected (Tab 2).

Tab 2. Yeast two-hybrid analysis of the interactions between the α_1A-AR C-terminus and the three cloned protein segments.

Cotransformants	HIS+	MEL1+	Growth on QDO plates
pGBKT7 + BMP-1/pACT2	–	–	–
pGBKT7 + Abr/pACT2	–	–	–
pGBKT7 + filamin-C/pACT2	–	–	–
a1A-AR-CT/ pGBKT7+pACT2 (1A-T)	–	–	–
a1A-AR-CT/pGBKT7+BMP-1/pACT2	+	+	+
a1A-AR-CT/pGBKT7+Abr/pACT2	+	+	+
a1A-AR-CT/pGBKT7+filamin-C/pACT2	+	+	+

HIS⁺, the reporter gene HIS was activated; MEL1⁺, the reporter gene MEL1 was activated; QDO, SD/-Leu-His-Ade-Trp selection culture.

Identification of the interactions between the α_1B-AR-CT or α_1D-AR-CT and the three cloned protein segments In order to identify whether the three protein segments interacted with the α_1A-AR subtypes specifically or not, the interactions between the other two α₁-AR-CTs and the three cloned protein segments were further tested. As performed for the α_1A-AR, we co-transformed BMP-1/pACT2, Abr/pACT2, and filamin-C/pACT2 with α_1B-AR-CT/pGBKT7 and α_1D-AR-CT/pGBKT7, respectively. Strikingly, both α_1B- and α_1D-AR C-terminal domains showed positive interactions with filamin-C, whereas neither of them interacted with BMP-1 or Abr. Co-transformation of α_1B-AR-CT/pGBKT7 or α_1D-AR-CT/pGBKT7 with pACT2 alone (1B-T or 1D-T) was not able to activate the yeast reporter genes and was used as control (Tab 3).

Tab 3. Yeast two-hybrid analysis of the interactions between the α_1B-AR or α_1D-AR C-terminus and the three cloned protein segments.

Cotransformants	HIS+	MEL1+	Growth on QDO plates
a1B-AR-CT/pGBKT7+pACT2 (1B-T)	–	–	–
a1B-AR-CT/pGBKT7+Abr /pACT2	–	–	–
a1B-AR-CT/pGBKT7+BMP-1/pACT2	–	–	–
a1B-AR-CT/pGBKT7+filamin-C/pACT2	+	+	+
a1D-AR-CT/pGBKT7+pACT2 (1D-T)	–	–	–
a1D-AR-CT/pGBKT7+Abr/pACT2	–	–	–
a1D-AR-CT/pGBKT7+BMP-1/pACT2	–	–	–
a1D-AR-CT/pGBKT7+filamin-C/pACT2	+	+	+

HIS⁺, the reporter gene HIS was activated; MEL1⁺, the reporter gene MEL1 was activated; QDO, SD/-Leu-His-Ade-Trp selection culture.

X-Gal and ONPG assay In the following experiments, we performed the co-transformation of different bait and prey constructs into the yeast Y187 cells carrying the β-galactosidase reporter genes to further confirm the positive reactions. The activity of β-galactosidase was measured qualitatively by the filter assay (X-Gal assay) and quantitatively by liquid culture assay (ONPG assay).

X-Gal assay In this assay, at least three independent clones were tested and all the positive co-transformants developed weak or strong blue color on the filter within about 6 h at 37 ºC, among which co-transformants of α_1A-AR-CT/pGBKT7 and BMP-1/pACT2 (1A-B) showed strong interactions (clones turned deep blue at about 30 min) while other positive transformants (α_1A-AR-CT/pGBKT7 and filamin-C/pACT2, 1A-F; α_1A-AR-CT/pGBKT7 and Abr/pACT2, 1A-A; α_1B-AR-CT/pGBKT7 and filamin-C/pACT2 , 1B-F; α_1D-AR-CT/pGBKT7 and filamin-C/pACT2, 1D-F) only showed weak but apparent blue at about 5-6 h, suggesting relatively weak interactions occurred (Fig 1). This assay was performed for more than three times.

Fig 1. X-Gal assay of interactions between three α₁-AR C-terminal tails and the three identified protein segments in yeast Y187 cells. (A) The interactions between α_1A-AR-CT/pGBKT7 and pACT2 alone (1A-T), filamin-C/pGBKT7 (1A-F), Abr/pGBKT7 (1A-A), or BMP-1/pGBKT7 (1A-B). The blue colors on the filter were recorded at 6 h after the transformants were transferred onto the filter and kept at 37 ºC. (B) and (C) indicated the results of the two methods carried out for the α_1B-AR-CT/pGBKT7 and α_1D-AR-CT/pGBKT7, respectively.

ONPG assay In this assay, the release of o-nitrophenol from the substrate ONPG was recorded at 420 nm and normalized to cell density measured photometrically at 600 nm; the β-galactosidase activity was presented in Miller units. Quantitatively, the interaction between α_1A-AR-CT and BMP-1 protein segment (1A-B) was about 30 times stronger than that of the control (1A-T) (P<0.01), while β-galactosidase activity of 1A-F or 1A-A were only about 4-5 times as strong as that of the control (P<0.05) (Fig 2A). β-galactosidase activity of 1B-F (P<0.05) or 1D-F (P<0.01) was about 2-3 times stronger than the control respectively (Fig 2B and 2C). The results obtained by the two methods were fully compatible.

Fig 2. Plasmids carrying the bait and constructed prey protein segments were co-introduced into yeast Y187 by the lithium acetate method. Transformants of pACT2 alone and pGBKT7 fused with each of the a-AR C-terminus were used as controls (1A-T, 1B-T, and 1D-T). The β-galactosidase activity in each transformant was monitored by the liquid ONPG assay and shown in miller units. At least three independent transformants were carried out in ONPG assay. Mean±SD. (A) The interactions between α_1A-AR-CT/pGBKT7 and pACT2 alone (1A-T), filamin-C/pGBKT7 (1A-F), Abr/pGBKT7 (1A-A), or BMP-1/pGBKT7 (1A-B). (B,C) The two methods carried out for the α_1B-AR-CT/pGBKT7 and α_1D-AR-CT/pGBKT7, respectively. ^bP<0.05, ^cP<0.01 vs control.

DISCUSSION

In this work, we identified novel interactions between the C-terminal tail of α₁-ARs and three non-G-proteins in the yeast two-hybrid systems. Of all the three proteins, α_1A-AR C-terminus showed a strong interaction with BMP-1 segments but interacted with filamin-C or Abr segments weakly, while the C-terminal tail of α_1B-AR or α_1D-AR only interacted with filamin-C weakly.

Yeast two-hybrid assay was an effective method for the identification of interaction between two proteins. Although it had the limitation that there maybe false-positives exist, compared with other methods, such as protein affinity chromatography, affinity blotting, immunoprecipitation and cross-linking, yeast two-hybrid assay had the advantage of screening all the protein-protein interactions in vivo, under which conditions the proteins observed were more likely to be in their native conformations^[7]. So, it has been widely used for the study of protein-protein interactions.

ARs were classical G-protein-coupled receptors (GPCR), alias, seven-transmembrane receptors. Recently, some reports came that the cytoplasmic C-terminal tails of ARs could interact with other proteins besides G-proteins. It meant that ARs could accomplish their biological functions in a G-protein-dependent or G-protein-independent way. Several proteins were found to interact with the C-terminal domain of ARs by yeast two-hybrid analysis. For instance, the a-subunit of eukaryotic initiation factor 2B (Eif-2b), a guanine nucleotide exchange protein that was involved in regulation of translation, was observed to associate with the C-terminal cytoplasmic domains of α_2A- and α_2B-AR in a yeast two-hybrid screen of a cDNA library prepared from HEK293 cell^[8]. In the following years, gC1q-R, a multifunctional protein, and PSD-95, a multiple PDZ domain-containing scaffolding protein, were identified as the binding partner of α_1B- and b₁-AR C-terminal tails respectively^[2,3].

Of all the subtypes of AR, α₁-AR, especially the α_1A-AR subtype, played a potential important role in the physiological or pathological processes, including the cell growth regulation, cardiac inotropy, hypertrophy, and so on. Considering the important roles that α_1A-AR played, it was imperative to find possible partners that bound to it and so that may affect its functions. In our study the C-terminal tail of α_1A-AR was used as a bait to screen a human brain cDNA library with an expectation to find novel proteins that might interact with α_1A-AR through its C-terminal domain. Fortunately, we found three novel and interesting proteins interacting with the α_1A-AR C-terminal domain in the yeast cells.

One of the proteins identified in our experiment is bone morphogenetic protein-1 (BMP-1), a zinc metalloproteinase whose metalloproteinase domain shared 39 % sequence identity with that of astacin, the digestive proteinase from crayfish^[9]. It was increasingly apparent that in addition to procollagen processing, BMP-1 could cleave several other substrate^[10-12] and thus played other roles central to extracellular matrix formation and early morphogenesis like embryo ventralization^[13-15]. Besides, BMP-1 was reported to process the homotrimer of type V procollagen^[16]. Struc-turally, the domains in BMP-1 included an N-terminal astacin-like zinc-binding metalloendopeptidase domain followed by varying numbers of epidermal growth factor-like (EGF-like) motifs and internal repeats termed CUB domains that might mediate binding to other extracellular proteins^[17-19]. However, until now, few intracellular proteins were reported to interact with BMP-1, let alone α_1A-AR. We detected a strong positive interaction (the activity of β-galactosidase was about 300 times stronger than the control) between the fifth CUB domain of BMP-1 and α_1A-AR, which indicated there might exist strong or significant protein-protein interactions between α_1A-AR and BMP-1 in eukaryotic cells. In fact, we confirmed the interaction between BMP-1 and α_1A-AR by ELISA assay in HEK293 cells ^[20]. Based on our present findings, we supposed that there might exist intracellular interactions between α_1A-AR and BMP-1 before it was secreted outside of the cell, which might lead to novel functional findings of the two proteins, such as α_1A-AR cell signaling or BMP-1 secretion.

The second protein was filamin-C, which belonged to the filamins family of high molecular mass cytoskeletal proteins that organize filamentous actin in networks and stress fibers. In humans three filamin genes were cloned: FLNA, FLNB, and FLNC, encoding three filamin proteins (filamin-A, B, and C) respectively. Different from other two filamins, filamin-C exists only in cytoskeletal muscles and myocardiac muscles. Further more, structurally, filamin-C assembled into tail-to-tail, non-covalently associated dimers. Their amino-termini encode an ABD (actin binding domain), which was followed by a rod-domain composed of 24 100-residue repetitive segments including a carboxy-terminal dimerization domain. It was clear that there existed an 81 amino acid insertion in repeat 20, which was not present in filamin-A or -B. So far, 20 plus proteins were found to bind to filamins, and more were expected^[21]. To this end, filamins were proposed to integrate cell mechanics as well as cell signaling. They not only interacted with peripheral actin networks but also bound to a variety of other ligand proteins through their C-terminal β-sheet repeats. Among those identified binding partners, six of them bound specifically to filamin-C C-terminal regions, which had been identified as γ,δ-sarcoglycan, androgen receptor, FATZ, myotilin, LL5b and PKCa^[22-27]. We also observed interactions among part region of filamin-C C terminus (repeats 24) and the C-terminal domain of the three subtypes of α₁-AR, which indicated that filamin-C played important roles in α₁-AR cellular localization and cell signaling. It was very interesting to find that filamin-C segments could nonspecifically interact with each of the C-terminal domain of the three α₁-AR subtypes, but whether these nonspecific interactions also existed in mammalian cells and whether their interactions played different roles still need to be further studied.

The other protein identified in our work was Abr, encoded by the active BCR-related gene (ABR). Interestingly, the Bcr-related protein Abr contained both Dbl- and GAP-homology domains analogous to those in Bcr, suggesting that it also might function as a bi-directional modulator of Rho family function^[28-31]. Until now, the molecular mechanisms underlying the Rho GTPases functioning were still not very clear. Using library screening, we detected that the C-terminus of α_1A-AR could bind to the C-terminal region of Abr, which might function as a bi-directional modulator of Rho family and had never been reported to bind to α_1A-AR.

In conclusion, by yeast two-hybrid assay, we found three novel protein partners of the C-terminus of α₁-AR. Although all the interactions were found only in yeast cells and took place just between parts of the proteins, it was sure that there indeed existed interactions between the three proteins and the C-terminal tails of the receptors. Besides, no matter strong or weak, the interactions were observed in vivo. This greatly enhanced the possibility that the interactions might also exist between the entire proteins under natural conditions and might be of great biological significance. However, other methods such as GST-pull down assay or co-immunoprecipitation assay should be done to further confirm their interactions in mammalian cells. Anyway, considering the physiological functions of the three proteins, we had the reason to expect that our findings in the yeast cells might provide new clues for the study of functional relationships between them and α₁-AR. We also demonstrated that yeast two-hybrid assay was really a useful method for detection of protein-protein interactions, and we were sure that more proteins were to be identified to bind with ARs using this method.

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