Molecular Vision 2001; 7:222-227 <http://www.molvis.org/molvis/v7/a31/> Received 7 May 2001 | Accepted 28 September 2001 | Published 1 October 2001

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Cloning and characterization of three salamander retinal G-protein beta subunits

James C. Ryan, Rosalie K. Crouch, Jian-xing Ma
 
 

Department of Ophthalmology, Medical University of South Carolina, Charleston, SC

Correspondence to: Jian-xing Ma, M.D., Ph.D., Department of Ophthalmology, Medical University of South Carolina, 167 Ashley Avenue, Charleston, SC, 29403; Phone: (843) 792-3206; FAX: (843) 792-1723; email: majx@musc.edu

Abstract

Purpose: Recent evidence has shown that the beta-gamma dimers (bg) of activated heterotrimeric G-proteins are important in many cellular signaling pathways. Since two distinct transducin alpha subunits have been cloned from the salamander retina, we aimed to identify and characterize the G-protein beta (Gb) subunits that are involved in visual signal transduction in the salamander.

Methods: A salamander retina cDNA library was screened using degenerate oligonucleotide primers designed from a compilation of known Gb sequences. Tissue specific expression was determined by reverse transcriptase PCR (RT-PCR).

Results: The library screening resulted in the cloning of three full-length sequences, two of which encode proteins of 340 residues and the third being an iniation variant of 353 and 395 residues. No identical matches were found in GenBank but each shows highest homology to G-beta-1 (b₁), G-beta-3 (b₃), and G-beta-5 (b₅ and b_5L) subunits of other species, respectively. The b₁ and b₃ subunits are 84.7% identical to each other but both show only 52% identity to b₅ at the protein level. RT-PCR analysis showed that all the subunits are expressed in multiple tissues, including the retina. However, the b_5L splice variant was found only in the retina.

Conclusions: Three distinct Gb subunit transcripts are expressed in the salamander retina. These subunits have proven to be important in the visual system of mammalian models.

Introduction

Most vertebrate retinas contain two distinct types of photoreceptors, rods and cones, with each type containing a major visual pigment in its outer segment. These visual pigments utilize a covalently bound chromophore ligand, 11-cis retinal or 11-cis 3,4 dehydroretinal [1]. Upon exposure to light, the chromophore absorbs a photon and isomerizes to its all-trans configuration [2]. This change causes a shift of the opsin's transmembrane helices, resulting in activation of the G-protein transducin [3]. The activated transducin complex dissociates into an a monomer and a bg dimer [3], and the ensuing phototransduction cascade produces a hyperpolarization of the photoreceptor. The function of the transducin a subunits (a_t) is well documented, however the role(s) of the associated bg dimers in the photoreceptor have not been as well studied. Nevertheless, it has been shown that neither the a_t nor its bg dimer effectively interacts with rhodopsin on its own, but requires a concerted effort to trigger visual signal transduction [4].

The mammalian rod and cone pigments interact with two distinct transducins, rod and cone transducins [5]. Until recently, there had only been a single transducin found in both frog (L07771) and fish [6] retina with the fish transducin showing intermediate homology to mammalian a_t1 and a_t2. The two newly cloned salamander transducin alpha subunits were found to reside in different photoreceptor cell types but it was still unknown if there existed distinct Gb subunits in the retina [7]. The salamander is a staple for electrophysiology models in vision research and as such has been the model for the elucidation of many aspects of the visual signal transduction process. This fact makes it important to clarify and compare the primary sequences of the salamander phototransduction proteins.

The Gbs belong to a large family of proteins with a WD (Tryptophan-Aspartate) repeating motif, which are distinguished by the recurrence of the WD-repeat from four to eight times. This motif is usually around 40 residues long and most are flanked by an N-terminal GH (Glycine-Histidine) and a C-terminal WD. The seven WD repeats of transducin's b subunit form a structure known as a b-propeller with the inner blades forming a water filled tunnel [8]. The bg dimer appears to undergo little to no conformational change upon activation of the heterotrimer [9]. However, there has been evidence to show the dimer to undergo structural change when bound to phosducin [10].

Phosducin is one of the key proteins that bg dimers interact with in the photoreceptor and in other cells. Phosducin closely interacts with the "top" surface of the b-propeller, which is also the face that interacts with the alpha subunit of transducin [9,11]. This could regulate heterotrimer activity by acting as a negative regulator of the bg dimer. Furthermore, it was also shown that GRKII (G-protein coupled receptor kinase II) phosphorylates the phosducin family on the C-terminal domain, markedly reducing bg dimer binding to phosducin, suggesting another possible point of pathway control [12].

Additional phototransduction proteins, rhodopsin, three cone pigments and two distinct arrestins, have also been cloned from the tiger salamander retina [13-16]. Of additional interest, we recently found the blue sensitive cone pigment to be identical to the pigment expressed in green rods of the salamander (unpublished data). Although these two types of cells have identical absorbance wavelength maxima, they display different physiological properties. This makes the remaining, undisclosed visual pigment interacting proteins an even more intriguing issue. We report here the cloning and localization of three Gb subunits from the salamander retina.

Methods

Animals and tissues

Care, use, and treatment of all animals in this study were done in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

PCR amplification of Gb cDNA fragments from a retina cDNA library

To obtain specific sequences of retina specific Gbs to be used as a probe, fragments of the Gb cDNA were first amplified from an aquatic phase salamander retina cDNA library (a gift from Dr. J. L. Arriza at Oregon Health Science University, Portland, OR) by PCR. This retina cDNA library was constructed in the l ZAP II phage vector (Stratagene, La Jolla, CA). Degenerate oligonucleotide primers GB-D and GB-U (Table 1), nucleotides 660-678 and 1307-1327 of salamander b₁, respectively, were synthesized according to the consensus sequences of Gbs from other species. The b₅ primers, GB5D-14 and GB5U-341, were designed from the mouse sequence of Gb₅. The PCR reaction mixture was as follows: 45 ml Platinum PCR Supermix (Gibco BRL Life Technologies, Rockville, MD), 2 ml each of 10 mM of 5' and 3' Gb consensus primer and 1 ml of 100 ng/ml salamander retina library in pBluescript. Low stringency PCR conditions were 30 cycles of 95 °C for 1 min, 50 °C for 1.5 min, and 72 °C for 2.5 min. Fragments were analyzed by agarose gel electrophoresis and then sequenced by dideoxy methods using an ABI automatic sequencer. FASTA searches of the GenBank database were performed to determine if the cloned sequences were of the Gb family. Based on the sequences of these Gb cDNA fragments, b₁, b₃, and b₅ specific probes were designed to screen a salamander retina cDNA library to clone the full length Gbs.

Positive selection screening of a cDNA library

The Arriza salamander retina cDNA library was screened using the GeneTrapper cDNA Positive Selection System (Gibco BRL) according to the manufacturer's protocol. The positive clones were sequenced using the ABI automatic sequencer.

Reverse transcriptase polymerase chain reaction (RT-PCR)

The retina, heart, brain, and skeletal muscle of 10 salamanders were dissected and single step RNA isolation was carried out with TRIzol reagent (Gibco BRL) according to manufacturers protocol. The RNA was quantified by absorbance spectroscopy at 260 nm. Total RNA (2 mg) was subjected to reverse transcription with M-MLV (Moloney Murine Leukemia Virus) Reverse Transcriptase (Gibco BRL) using random nonamers (Sigma) as per the Gibco BRL M-MLV protocol. The RT product (2 ml) was amplified by PCR using either b₁, b₃, b₅ specific or GAPDH (glyceraldehyde phosphate dehydrogenase) primers (Table 1). The PCR reaction mixture was as follows: 45 ml Platinum PCR Supermix (Gibco BRL), 1.5 ml each of 10 mM 5' and 3' Gb specific primer and 2 ml of RT template. PCR reaction conditions were as follows: 90 s at 94 °C for 1 cycle and 80 s at 94 °C, 80 s at 56 °C s, and 95 s at 72 °C for 30 cycles. The PCR product (10 ml) was analyzed by electrophoresis on a 1% agarose gel.

Results

Cloning and sequencing of the salamander Gb cDNAs

Using Gb specific probes, Gb₁, Gb₃, and Gb₅ subunits were isolated from 2 x 10⁶ pfu of the cDNA library of the salamander retina. Seventeen Gb₁, 23 Gb₃, and 11 Gb₅ clones showing positive colony hybridization results were purified and digested with EcoRI. The colonies with the largest inserts were sequenced, resulting in full length coding regions. The Gb₁ cDNA (GenBank accession AF277161) is 1619 base pairs (bp) in length, consisting of 313 bp of 5' untranslated region (UTR), 1023 bp of coding sequence, and 283 bp of 3' UTR. The first ATG (position 314), in the context of a Kozak sequence [17], is designated as the translation start codon and the termination codon TAA was identified at position 1334. A poly-A tail was identified without the typical preceding polyadenylation signal, AATAAA.

The Gb₃ cDNA (GenBank accession AF277162) is 1766 bp in length, including 202 bp of 5' UTR, 1023 bp of coding region and 541 bp of 3' UTR. The translation start codon ATG in a Kozak sequence is located at position 203 and the termination codon, TGA, is located at position 1223. No typical polyadenlylation signal or poly-A tail was identified in the sequenced clones.

The Gb₅ cDNA (GenBank accession AF369757) initiation variant is 1775 bp in length consisting of 46 bp 5' UTR, 1185 bp of coding sequence, and 544 bp of 3' UTR. The first ATG, position 47, is designated as the translation start codon for the long variant. The second ATG in the same ORF, position 173, is designated as the start codon for the shorter variant. The termination codon, TAA, was identified at position 1232. No poly-A tail was identified.

Deduced amino acid sequence of Gb cDNAs: The Gb₁ cDNA encodes a protein of 340 amino acids (Figure 1). As calculated by GCG software, the deduced protein has a molecular weight of 37.33 kDa, and an isoelectric point of 5.86. The deduced Gb₃ sequence consists of 340 amino acids with a molecular weight of 37.35 kDa and an isoelectric point of 6.29. The deduced Gb₅ sequence consists of 353 amino acids with a calculated molecular weight of 38.86 kDa and an isoelectric point of 5.97. The Gb_5L variant contains 395 residues, has a molecular weight of 43.42 kDa and an isoelectric point of 6.28.

A global GenBank search using the GCG program FastA at default settings showed that these salamander Gbs do not match any existing sequences but have significant sequence similarities to other Gb subunits. The Gb₁ peptide shares high sequence identity to Gb₁ from other species, 98.5% to frog and 96.2% to human, but relatively lower identity (82.9%) to the human Gb₃. In contrast, the Gb₃ is 89% identical to the mammalian Gb₃s but has relatively lower identity, 83.5%, to the mammalian Gb₁ sequences. The salamander Gb₅ subunit shows distant identity to all of the other four human beta subunits, approximately 50%, but 96% to the human b₅. Phylogenetic analysis of the Gb families placed our clones firmly in the class of either Gb₁, Gb₃ or Gb₅ (Figure 2). The peptide sequences of the seven potential WD repeat domains for all three beta subunits were aligned and compared (Figure 3). The highly conserved aspartic acid between strands b and c was present in all the repeats of all the subunits.

RT-PCR

To investigate tissue distribution of these Gbs, we performed RT-PCR with sequence specific primers for each of the three subunits (Table 1) in the brain, heart, skeletal muscle, and retina (Figure 4). Both b₁ and b₃ subunits were found in all the tissues analyzed. Transcripts of the b₅ subunit were found in the brain, heart, and retina. The b_5L transcript was found only in the retina (Figure 4).

Discussion

Visual signal transduction has long served as the paradigm for G-protein coupled receptor (GPCR) study. In fact, the rhodopsin-transducin interaction is the best characterized of all GPCR systems with the salamander model used extensively for this research. However, the molecular investigation lags far behind the efforts into the biochemical and physiological aspects. There was only a single known Gb sequence of an amphibian species in GenBank, Xenopus b₁ [18]. We felt that it was important to clarify the molecular details. This study represents the first evidence of distinct Gb sequences residing in the amphibian retina.

Three Gb subunits have been cloned from a salamander retina library that show significant homology to b₁, b₃, and b₅ sequences of other species. Phylogenetic analysis has decisively classified these subunits into the families of their highest respective sequence homologies (Figure 2). The Gb subunits have been shown to be part of the amplification phase of phototransduction in rod and cone photoreceptors of other species with b₁ being expressed in rods but not cones and b₃ being expressed in cones but not rods [19].

RT-PCR has shown the presence of the b₁ and b₃ subunit transcripts in the retina, heart, muscle, and brain. This is not surprising since these Gb subunits are expressed nearly ubiquitously throughout the body in other species. Preliminary immunohistochemistry using a commercial b₁ antibody (Calbiochem, La Jolla, CA, catalog number 371813; data not shown), consistent with mammalian models, showed staining in the outer segments of rods but not in the cones (staining for b₃ was not attempted due to the lack of epitope identity between bovine (commercial) and salamander subunits). RT-PCR showed the b₅ subunit was found in the salamander retina, heart, and brain. Initially, Watson et al. [20], showed in the mouse that the b₅ transcript is expressed primarily in the brain. Later, Jones et al. showed that in humans the transcript is expressed predominantly in the brain, pancreas, heart, and kidney with lower levels in other tissues as well [21]. The previous identification of tissue specificity in the mouse and human was done by Northern blot analysis. Although less prone to artifacts, Northern blotting is not as sensitive as PCR, and thus, tissue distribution for the b₅ could be more extensive than previously thought. With 30 cycles of amplification, no RT-PCR product from salamander skeletal muscle was detectable under our conditions. However, when the PCR amplification cycles were increased to 36, a faint product was visible in skeletal muscle, suggesting a low level expression. Additionally, the b_5L transcript was found only in the retina, as previously described in the mouse [22].

At the protein level, the salamander Gb subunits show the same characteristic seven repeats of the WD-40 motif as seen in all Gb subunits. Some functions of WD repeat proteins include signal transduction, RNA processing, gene regulation/development, vesicular traffic, and regulation of cytoskeletal assembly [23]. The single unifying theme of the WD repeat family is their ability to act as a scaffolding protein, allowing other proteins to interface [23,24]. The repeats are simply numbered according to their position in the gene sequence. The seven repeats are known to have higher homology between the same repeat position in other species than among the repeats of different positions in the same protein (Figure 3). For example, the salamander Gb₁ first repeat shows greater homology to the first repeat of the bovine Gb₁ sequence than to any of the other six salamander repeats. The greatest conservation in the repeating unit is the aspartic acid between strands b and c. This residue is strictly conserved in all the repeats of the three salamander Gb subunits. The residues in repeat 5 have been shown to confer selectivity for binding the g subunit [25-27]. Since mammalian systems have been found to use the dimer pairs b₁g₁ for rod and b₃g_{8_} for cone, there must be a region where the homology is low. In the salamander subunits, there is one stretch of residues in repeat 5 that could possibly offer such selectivity, residues 239-242. Three of the four residues are very different between the b₁ and b₃ subunits (Figure 1).

Liu et al. [28] discovered a conserved calmodulin binding site on the N-terminal of the Gb subunit that, when occupied, interfered with heterotrimer formation but had little effect on phospholipase Cb₂ activity. Residues 43-60, which partially overlap the first WD repeat, and specifically, Ile-43, Arg-48, Arg-49, Leu-51, and Leu-55 were established to be central. We found these residues strictly conserved in the salamander b₁ and b₃ subunits but only Arg-48, Arg-49, and Leu-51 conserved in b₅, suggesting that Ca²⁺ may regulate their activities in the salamander to some degree.

Zhang et al. [29] recently identified the Gb₅ in complex with RGS7 (regulator of G-protein signaling), in membrane, cytosolic, and nuclear fractions of PC12 cells as well as mouse brain nuclear extract. It is also documented that the Gb_5L subunit in the retina strongly associates with RGS9. In fact, the RGS9 knockout mouse showed the absence of Gb₅ at the protein level and prolonged recovery to light stimuli [30]. The diverse locations of the Gb₅ may be indicative of a more ranging functionality.

Phosducin interactions with bg dimers have been reported to block interactions of the dimer with the Ga and thusly the trimer's interaction with rhodopsin [31]. It was shown by x-ray crystallography that the conformation of the b₁ and b₃ dimer shifts slightly between residues 287-295, 308-318, and 329-338 when bound to phosducin [10]. In the salamander Gb₁ and Gb₃, residues 299-305 mark the region of least identity in the entire protein while residues 306-319 mark the beginning of the conserved seventh WD repeat.

The salamander has been used extensively for physiological studies of the visual transduction process and many significant findings have been reported based on this model. Previously, there was no record of two distinct transducins in lower vertebrates, and it was uncertain whether these G-proteins had yet diverged in amphibians. In fact, there was a single transducin cloned from fish that showed similar homology to both rod and cone transducins from mammals [6]. Recently, the salamander was shown to express photoreceptor specific Gas, rod and cone transducins [7]. Since the salamander is used extensively for vision research, it is imperative that we analyze the visual signal transduction components on a molecular level. The recently proven high sequence identity between human and salamander G-proteins subunits allows us to better apply knowledge gained from this animal model to human vision.

Acknowledgements

This study was supported by grants from National Science Foundation MCB9600772 and National Institute of Health EY12231 (J. Ma), a grant from National Institute of Health EY04939 (R.K. Crouch) and an unrestricted grant to the Department of Ophthalmology, MUSC from Research to Prevent Blindness, Inc.

Sequence analysis was accomplished via GCG software licensed by the Biomolecular Computing Resource, an element of MUSC's University Research Resource Facility.

The template for generating the WD repeat pattern was produced at Boston University, http://bmerc-www.bu.edu/bioinformatics/wdrepeat.html

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