Identification of Sphingolipid-binding Motif in G Protein-coupled Receptors 10 Sandeep Shrivastava, Md. Jafurulla, Shrish Tiwari, and Amitabha Chattopadhyay Abstract report on the presence of SBM in serotonin1A Sphingolipids correspond to a major class of receptors and provide novel insight on the lipids which serve as indispensable structural molecular mechanism of GPCR-sphingolipid components of membranes and play an impor- interaction. tant role in various cellular functions. They constitute ~10–20% of total membrane lipids Keywords and are known to form segregated domains in SBM · Sphingolipids · GPCR · Serotonin1A biological membranes. Sphingolipids have receptor · CRAC been shown to play a vital role in the function of various G protein-coupled receptors (GPCRs). We report here the presence of sphingolipid- binding motif (SBM) in representative GPCRs Abbreviations such as cholecystokinin, oxytocin and secretin receptors, and subtypes of human serotonin CRAC Cholesterol recognition/interaction receptors. We previously reported the impor- amino acid consensus tance of sphingolipids in the function of the GPCR G protein-coupled receptor SBM Sphingolipid-binding motif serotonin1A receptor, a representative member of the GPCR superfamily, involved in behav- ioral, cognitive, and developmental functions. In this work, we show that the serotonin 1A 10.1 Introduction receptor contains a putative SBM, corres- ponding to amino acids 205 to 213. In addition, Sphingolipids are indispensable constituents of our analysis shows that SBM is an intrinsic cellular membranes of eukaryotes and account characteristic feature of the serotonin recep- 1A for ~10–20% of the entire lipids associated with tor and is conserved throughout the course of membranes (Holthuis et al. 2001). They are natural evolution. Our results represent the first believed to form laterally segregated domains with cholesterol (also referred as ‘lipid rafts’) S. Shrivastava · M. Jafurulla · S. Tiwari (Brown 1998; Masserini and Ravasi 2001; A. Chattopadhyay (*) Jacobson et al. 2007). The concept of these mem- CSIR-Centre for Cellular and Molecular Biology, brane domains assumes relevance as they have Hyderabad, India e-mail: [email protected]; [email protected]; been implicated in crucial physiological functions [email protected]; [email protected] such as cellular sorting, trafficking (Simons and # Springer Nature Singapore Pte Ltd. 2018 141 K. Chattopadhyay, S. C. Basu (eds.), Biochemical and Biophysical Roles of Cell Surface Molecules, Advances in Experimental Medicine and Biology 1112, https://doi.org/10.1007/978-981-13-3065-0_10 142 S. Shrivastava et al. van Meer 1988), cellular signaling (Simons and variety of membrane proteins has been attributed Toomre 2000), and the entry of pathogens into to specific interaction (Snook et al. 2006). host cells (Riethmüller et al. 2006; Pucadyil and A characteristic amino acid sequence which Chattopadhyay 2007; Vieira et al. 2010; could represent the conserved sphingomyelin- Chattopadhyay and Jafurulla 2012; Kumar et al. binding motif in proteins has been previously 2016). proposed (Contreras et al. 2012). These authors The G protein-coupled receptor (GPCR) super- identified a specific binding motif for family constitutes an important class of proteins sphingomyelin, termed the sphingolipid-binding employed in signal transduction (Rosenbaum et al. motif (SBM), in the transmembrane protein p24 2009; Chattopadhyay 2014). GPCRs are (a COPI machinery protein) and demonstrated the associated with an array of physiological processes headgroup and acyl chain specificity of and have therefore emerged as major drug targets sphingomyelin in its interaction with this motif. (Heilker et al. 2009;Chattopadhyay2014;Cooke Importantly, these authors further showed that et al. 2015;Jacobson2015). An estimate of ~50% only sphingomyelin with an appropriate dynamic of present clinically recommended drugs act as volume would fit into the cavity formed by SBM either agonists or antagonists to GPCRs (Jacobson residues in the transmembrane domain of p24. In 2015). The serotonin1A receptor represents an spite of the importance of sphingolipids in the important G protein-coupled neurotransmitter structure and function of several membrane receptor and plays a vital role in several neurolog- proteins, including GPCRs and ion channels ical functions such as behavior, cognition, anxiety, (Alves et al. 2005; Harikumar et al. 2005; Sjögren depression, and learning (Pucadyil et al. 2005; and Svenningsson 2007; Fantini and Barrantes Müller et al. 2007; Kalipatnapu and 2009; Slotte 2013; Jafurulla and Chattopadhyay Chattopadhyay 2007; Fiorino et al. 2014). It there- 2015; Jafurulla et al. 2017), limited information is fore naturally has emerged as an important target available on specific binding motifs involved in in the development of therapeutics against neuro- GPCR-sphingolipid interaction. Exploring the logical disorders (Kaufman et al. 2016). possibility of specific interaction between GPCRs are transmembrane proteins, and a GPCRs and sphingolipids therefore assumes rele- large part of the receptor remains embedded in vance. In the present study, we identified the the membrane. Any change in the membrane presence of SBM in representative GPCRs such lipid milieu therefore could influence the structure as cholecystokinin, oxytocin and secretin and function of the receptor. For example, it is receptors, and subtypes of human serotonin estimated from molecular dynamics simulations receptors. Earlier results from our laboratory that the lipid-protein interface accounts for ~38% have shown that membrane sphingomyelin is of the entire surface area of rhodopsin (Huber et al. important for regulating the function of the 2004). Keeping in mind the increasing relevance serotonin1A receptor (Jafurulla et al. 2008; Singh of the serotonin1A receptor in pharmacology and and Chattopadhyay 2012). We therefore explored drug development, interaction of surrounding the presence of SBM in the human serotonin1A membrane lipids with the receptor assumes signif- receptor and its conservation over the course of icance. In this context, previous work from our natural evolution. Our results show that human laboratory has demonstrated the requirement of serotonin1A receptors contain a putative SBM in membrane cholesterol and sphingolipids transmembrane helix V. In addition, sequence (reviewed in Pucadyil and Chattopadhyay 2006; analysis of the serotonin1A receptor from various Paila and Chattopadhyay 2010; Jafurulla and species across diverse phyla shows that SBM is Chattopadhyay 2013, 2015) in the function of the an inherent characteristic attribute of the receptor serotonin1A receptor. The influence of and is conserved throughout the course of natural sphingolipids on the structure and function of a evolution. 10 Identification of Sphingolipid-binding Motif in G Protein-coupled Receptors 143 10.2 Methods analyzed by examining amino acid sequences of the receptor over various phyla obtained from 10.2.1 Identification of SBM NCBI and ExPASy databases (see Fig. 10.3). in Representative Human Partial, duplicate, and other non-specific GPCRs sequences were removed from the set of sequences obtained. Initial alignment of The conserved signature sequence for SBM is sequences was carried out using ClustalW. After (I/L/T/V)XX(I/L/T/V)(I/L/T/V)XX(I/L/T/V) eliminating the relatively divergent parts of the (F/W/Y), where X represents any of the 20 natu- receptor, the sequence was realigned using the rally occurring amino acids (Contreras et al. same program. The putative SBMs in the fi 2012; Björkholm et al. 2014). The putative serotonin1A receptor were identi ed by visual SBMs were identified by visual inspection in inspection. The amino acid sequences used for cholecystokinin, oxytocin, serotonin, and secretin the analysis belong to diverse taxa that include receptors with the help of regular sequence of insects, fish and other marine species, SBM (see Fig. 10.1a). The amino acid sequences amphibians, and extending up to mammals. The of GPCRs were obtained from NCBI database. quality of alignment shown in Figs. 10.1b and The positions of starting amino acid residues in 10.3 was computed in Jalview, the software the corresponding sequences are marked in used to view the alignment. parentheses. 10.3 Results and Discussion 10.2.2 Sequence Alignments Sphingolipids have been shown to modulate the of the Predicted SBM in Human function and organization of important classes of Serotonin Receptor Subtypes membrane proteins such as GPCRs (Jafurulla and Chattopadhyay 2015). In the overall context of Multiple sequence alignment for various seroto- sphingolipid sensitivity of GPCR function nin receptor subtypes was performed with (Sjögren and Svenningsson 2007; Fantini and ClustalW (Larkin et al. 2007), using the human Barrantes 2009; Paila et al. 2010; Singh et al. serotonin receptor sequence as a reference. The 1A 2012; Jafurulla and Chattopadhyay 2015; positions of starting amino acid residues in vari- Jafurulla et al. 2017), we examined whether the ous serotonin receptor subtypes are marked in sequence of some of the representative GPCRs parentheses (see Fig. 10.1b). includes any SBM(s). We identified the presence of SBM in representative GPCRs such as chole- cystokinin, oxytocin, serotonin1A, and secretin 10.2.3 Sequence Alignment receptors (see Fig. 10.1a). Interestingly, the func- of the Serotonin1A Receptor tion of some of these receptors (e.g., the chole- and Identification of SBM cystokinin receptor)