Visual arrestins in olfactory pathways of Drosophila and the malaria vector mosquito Anopheles gambiae C. E. Merrill*, J. Riesgo-Escovar†‡, R. J. Pitts*, F. C. Kafatos§, J. R. Carlson†, and L. J. Zwiebel*§¶ *Department of Biological Sciences, Program in Developmental Biology and Center for Molecular Neuroscience, VU Station B35-1812, Vanderbilt University, Nashville, TN 37235-1812; †Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520; ‡Department of Developmental Neurobiology, Centro de Neurobiologı´a,Apdo. Postal 1-1141, Quere´taro, Qro 76001, Mexico; and §European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany Edited by John H. Law, University of Arizona, Tucson, AZ, and approved November 7, 2001 (received for review September 24, 2001) Arrestins are important components for desensitization of G protein- transduction systems. Taken together this report establishes that coupled receptor cascades that mediate neurotransmission as well as particular insect arrestins are important components in both visual- olfactory and visual sensory reception. We have isolated AgArr1,an and nonvisual-signaling pathways. arrestin-encoding cDNA from the malaria vector mosquito, Anopheles gambiae, where olfaction is critical for vectorial capacity. Analysis of Experimental Procedures AgArr1 expression revealed an overlap between chemosensory and Generation and Screening of a Subtracted cDNA Library. Total RNA photoreceptor neurons. Furthermore, an examination of previously was prepared from hand-dissected olfactory tissue (antennae and identified arrestins from Drosophila melanogaster exposed similar maxillary palps) or carcasses (bodies stripped of heads including bimodal expression, and Drosophila arrestin mutants demonstrate antennae, maxillary palps, and all other appendages) of A. gambiae impaired electrophysiological responses to olfactory stimuli. Thus, we by using RNeasy kit (Qiagen, Chatsworth, CA). Selective amplifi- show that arrestins in Drosophila are required for normal olfactory cation via biotin and restriction-mediated enrichment (SABRE) physiology in addition to their previously described role in visual (11) was used to prepare an ‘‘antennae minus carcass’’ subtracted signaling. These findings suggest that individual arrestins function in library. The original protocol was modified by using SuperScript II both olfactory and visual pathways in Dipteran insects; these genes reverse transcriptase (GIBCO͞BRL) for first-strand cDNA syn- may prove useful in the design of control strategies that target thesis and by redesigning PCR primers. Purified antennal cDNAs olfactory-dependent behaviors of insect disease vectors. were amplified and subtracted an additional time or subcloned directly into pBluescript II-KS (Stratagene). he events that initiate and terminate chemosensory signal Initial sampling of 53 insert-containing plasmids by DNA se- Ttransduction have received considerable attention in recent quencing showed that eight cDNA fragments had significant sim- years. Olfactory transduction is mediated by G protein-coupled ilarly to arrestins by BLAST (12) analysis. One fragment was chosen receptor (GPCR) second messenger pathways (1, 2). Olfaction for further study based on its strong similarly to a 94-aa stretch of exhibits not only activation but also adaptation, whereby a progres- antennal-specific arrestins. Specific primers were designed for both Ј Ј sively weaker response is generated to repeated or persistent 5 and 3 rapid amplification of cDNA ends (RACE; ref. 13) by stimuli. At the molecular level, desensitization of GPCR signaling using a Marathon cDNA amplification kit (CLONTECH). These Ј Ј is brought about by reduced coupling between the receptor and were designated Arr5 -RACE (5 -CTAGTCTCCAGCGATGC- Ј Ј Ј heterotrimeric G proteins. After receptor phosphorylation by G CACTGTGTT-3 ) and Arr3 -RACE (5 -CAGCTGGGTGTGT- Ј protein-receptor kinases, binding of an arrestin uncouples GPCRs GGATGTGGTGC-3 ). Finally, the complete cDNA representing Ј Ј from the signaling cascade (3). Arrestins also have been shown to this A. gambiae arrestin gene was engineered by joining 5 and 3 be involved in GPCR internalization, an integral component of RACE products before final ligation into pBlueScript II (KS) GPCR resensitization in many systems (4). (Stratagene) and was designated AgArr1. Most arrestins characterized to date have been divided into two broad categories that are largely reflective of their presump- Sequence Analysis. Sequence comparisons with the DNA Data Base ͞ ͞ tive functional roles (5). Visual arrestins have been reported to in Japan European Molecular Biology Laboratory (EMBL) display nearly exclusive expression in photoreceptors and inter- GenBank and SWISSPROT databases were performed by using act with rhodopsin to terminate visual stimulation (6, 7). The the GCG software (14), and protein alignment was performed by second general arrestin subtype category comprises the non- using the CLUSTAL W software package (15). visual arrestins that are expressed in a wide variety of tissues (excluding photoreceptors) where they are presumed to interact Reverse Transcription–PCR (RT-PCR). RNA for developmental pro- with a diverse array of GPCRs (8). files was made as above by using whole bodies at various stages: (i) In light of the rapid progress in understanding the mechanisms embryos refer to freshly laid fertilized eggs; (ii) early larvae are 1–2 that govern olfaction (9), we have initiated molecular studies to instars, (iii) late larvae are 3–4 instars, and (iv) pupae refers to extend this understanding to insect disease vectors. Olfaction pupae taken at least 6 h before eclosion. Total adult A. gambiae principally mediates host preference, a trait that strongly contrib- RNA was prepared as described above from either female or male utes to the ability of Anopheline mosquitoes to act as vectors for malaria or other serious human diseases (10). We now report the This paper was submitted directly (Track II) to the PNAS office. cloning and characterization of AgArr1, a novel arrestin family Abbreviations: GPCR, G protein-coupled receptor; RACE, rapid amplification of cDNA ends; member from Anopheles gambiae sensu stricto (hereafter A. gam- EAG, electroantennogram; EPG, electropalpogram; EA, ethyl acetate; SH3, Src homology 3; biae). Additionally, we show its expression in both olfactory and BU, butanol; RT-PCR, reverse transcription–PCR. visual systems. Such an overlap in sensory system expression is Data deposition: The sequence reported in this paper has been deposited in the GenBank verified by studies of two arrestins in Drosophila melanogaster database (accession no. AY017417 for AgArr1). previously thought to be exclusively visual in nature; both Arrestin1 See commentary on page 1113. and Arrestin2 (DmArr1 and DmArr2) are expressed not only in ¶To whom reprint requests should be addressed. E-mail: [email protected]. photoreceptors, but also in olfactory neurons. By using well- The publication costs of this article were defrayed in part by page charge payment. This established olfactory paradigms in the Drosophila model system, we article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. NEUROBIOLOGY directly demonstrate the function of arrestins in olfactory signal §1734 solely to indicate this fact. www.pnas.org͞cgi͞doi͞10.1073͞pnas.022505499 PNAS ͉ February 5, 2002 ͉ vol. 99 ͉ no. 3 ͉ 1633–1638 Downloaded by guest on October 2, 2021 head appendages (antennae, palps, and proboscis), heads stripped SPOT camera system (Diagnostic Instruments, Sterling Heights, of appendages, or carcasses (bodies stripped of heads). PCRs were MI). Drosophila stainings were done as in ref. 19 by using Abs carried out with AgArr1 oligonucleotide primers ARR5.0 (5Ј- directed against DmArr1 and DmArr2 kindly provided by P. Dolph TTAAGGCCATGGTCCAGCAGGGTG-3Ј) and ARR-R5 (5Ј- (Dartmouth University, Hanover, NH) and C. Zuker (University of CGTTGCGTCGTCTATTCAAA-3Ј). Expression of ribosomal California, San Diego) (20). Sections and whole mounts were protein s7 (16) was assayed in parallel reactions by using sequence- incubated with horseradish peroxidase-conjugated secondary Abs, specific primers: S7a (5Ј-GGCGATCATCATCTACGTGC-3Ј) developed, mounted, and photographed by using a Zeiss Axiophot and S7b (5Ј-GTAGCTGCTGCAAACTTCGG-3Ј). D. melano- microscope. gaster RT-PCR reactions were performed on adult RNA generated as above from acetone-fixed, hand-dissected third antennal seg- Electrophysiology. Electrophysiological recordings of antennae and ments, heads stripped of antennae, and bodies with heads removed. maxillary palps in D. melanogaster were done with two different The reactions to amplify DmArr1 used primers DmArra1 (5Ј- setups, as described in refs. 19 and 21. To generate recordings, glass CAACTCCAACAAGGTGGTGA-3Ј) and DmArra3 (5Ј-GCT- microelectrodes were positioned on the surfaces of these olfactory TCCAGTTGGGCCTTG-3Ј); DmArr2 used primers DmArrb1 organs until electrical contact was made. Reference electrodes were (5Ј-ATGGTGAACGCCCAGTTTAG-3Ј) and DmArrb2 (5Ј- inserted into the head capsule. Most experiments were carried out GGCGAAGTCCTCGAATACAA-3Ј). PCR for DmKrz cDNA independently in both setups and gave similar results. Dilutions of was performed with krzL1 (5Ј-GGTGGTGGTGGAGGAAGTG- odorants were in paraffin oil. 3Ј) and krzR1 (5Ј-GCTGCTCACCGACTTTGGAT-3Ј) (17). In all reactions, primers for the ribosomal protein 49 gene (rp49a,