ReviewUrinary proteins and the modulation of chemical scents in mice and rats
Introduction
The presence of large amounts of protein in urine (proteinuria) is often considered to be a pathological condition, indicative of serious renal damage. While this is certainly the case in man, there are other species for which such a proteinuria is an obligatory condition. These urinary proteins were first observed in laboratory mice and rats, and it is the urinary proteins in these two species that have been most extensively understood. The proteins are known collectively as major urinary proteins (MUPs) or in the rat, as alpha 2u globulins (A2Us) (for recent reviews see [1], [2]). There is good evidence for MUPs also being expressed in glandular tissues such as salivary and mammary glands, nasal tissue and in the respiratory epithelia [3], [4], [5], [6] but we will not address the functions of these variants in this brief review.
Mice and rats deposit urine marks extensively around their territories (Fig. 1). Because scent marks are assessed in the absence of the depositing animal, the receiver must be able to glean a complex array of information from the scent signal – the sex, reproductive and social status of the donor, the time since the mark was deposited, and the identity and relatedness of the donor [7]. The receiver may also pick up information such as the health status of the donor [8], and the nature of the food they are consuming [9], [10]. The complexity of information embedded in scent marks must be conveyed by a complex mixture of chemicals. Some responses can occur with an airborne signal, others require physical contact between the receiver animal and the scent mark. Additional dimensions to the reception of a chemical signal include the time since the mark was deposited, and the pattern of scent marks that are distributed through the territory. Understanding the interplay between behavioral and biochemical factors in deposition and reception of scents is challenging, and many aspects of this interaction remain elusive.
It is increasingly clear that the proteinaceous component in mouse urine is critical to some aspects of scent communication. Urinary MUPs contain bound molecules that are pheromonally active, establishing beyond all dispute their role in delivering chemical signals. Any endeavor to understand the function of MUPs in chemical communication must therefore include stringently designed behavioral experiments using appropriately manipulated biochemical samples.
Scent marks, notably those deposited by a dominant animal, are encountered by conspecifics of both sexes and of different status. The effects of male urine scents on female conspecifics can be dramatic, and include such phenomena as pregnancy block and puberty acceleration [11], [12]. Moreover, there is good evidence that a female mouse uses the pattern and quality of male scent marks in selection of a mate [13], [14], [15]. Male conspecifics can be animals that are subordinate to the scent mark owner, in which case they will investigate and then tend to avoid the scent mark [16], [17]. In particular, subordinates will avoid countermarking with their own scent, depositing their urine instead in larger pools away from sites marked by the dominant male, presumably to avoid arousing attack from the dominant territory owner [18], [19]. Other males that are owners of neighbouring territories, or potential challengers to the current territory owner, will deposit their own urine countermarks (Fig. 2). This is the first skirmish in a ‘scent war’ [20] that is fought in part with chemical weapons – the scent marks themselves.
The protein concentration of wild-derived mouse urine is of the order of tens of milligrams per millilitre [21], which could be a substantial investment in protein synthesis that is lost irreversibly from the body. Additional costs associated with deposition of the marks include predation risk and the time and effort involved in continually refreshing scent marks throughout their territory. What is the evolutionary advantage to the mouse in incurring the cost of producing, disseminating and losing this protein? In this short review, we will summarize current knowledge of the biochemistry of MUPs, and show how the proteins can play multiple roles in the delivery of information in scent marks.
Section snippets
Structure of MUP and A2U
X-ray and NMR structures have been obtained for MUP, as have X-ray structures for A2U (Table 1). The structures are very similar, each having an eight-stranded beta-barrel fold that classifies them as lipocalins [27], and with a markedly similar structure (Fig. 3). A key feature of such lipocalins is a central cavity, lined with hydrophobic residues that constitute the site of binding of apolar ligands, especially pheromonal molecules (Fig. 4). In MUPs, this cavity has a volume of about 420–600 Å
Ligand binding
There had been intermittent reports in the literature that the pheromonal qualities of mouse urine could be delivered in part by a high molecular weight fraction. In many of these early studies, the high and low mass fractions were separated from each other by simple methods such as dialysis or by ammonium sulfate fractionation to precipitate the proteinaceous material. The association of the biological effects with this high mass fraction was taken as evidence that the pheromones were
Primary sequence polymorphisms in MUPs
MUPs recovered from the same inbred mouse strain consisted of several isoforms that could be resolved by electrophoresis or isoelectric focusing and indeed, co-inheritance of specific isoforms was used as a biochemical marker in genetics. Subsequently, the cloning of MUP cDNA sequences and the characterization of the gene structure confirmed that a region of mouse chromosome 4 encoded large numbers (over 30) of MUP genes and highly homologous but non-expressed pseudogenes in a complex
Sexual dimorphism in MUP expression
It is a widely held notion that MUPs are produced exclusively by sexually mature male mice. This may have derived from studies of inbred laboratory strains where the difference between the sexes is most marked. Also, some analyses have concentrated on the level of MUP mRNA present in liver of the subjects, rather than evaluating urinary output. A second complicating factor is the concentration of urine. Dilute urine would give a MUP concentration that is apparently less than that from an
The role of MUPs in mouse urine scent communication
Questions concerning the role that MUPs play in communication can only realistically be answered by combining biochemical studies with the assessment of behavioral and physiological responses to scent signals. Our experimental paradigm reflects a strong collaboration between biochemists and behaviorists, using natural responses to experimentally manipulated scent sources. Most of our research is targeted to wild-caught or wild-derived mice, as these exhibit a wider range of functionally
MUPs and histocompatibility complex (MHC)
Another highly polymorphic system that influences scent signals is the major histocompatibility complex, which is involved in self–non-self recognition at the cellular level in the immune system. MHC haplotype affects the scents produced by individuals in a wide range of species including rodents [66], [67]. Experiments utilizing MHC congenic strains of mice and rats have revealed that rodents can discriminate scents caused by single gene mutations in the MHC (e.g. [67], [68], [69], [70], [71]
MUP receptors?
There is an emerging body of evidence to support the idea that MUPs convey information in their own right, with or without the involvement of their bound ligands. However, the existence of specific receptors for MUPs remains somewhat controversial. The role of MUPs in mediation of pheromone signals strongly implicates the vomeronasal organ (for a recent comprehensive review of VNO see [87]). Certainly, there is now increasing evidence that a subclass of receptors (V1R) in the vomeronasal system
Conclusions
It is increasingly clear that the chemical signals in mouse urine elicit complex effects, and that multiple classes of molecules must be involved. Urine is such a critical source of complex information in mouse social behavior that it would be most unlikely that this role could be discharged by just a few compounds. It is now firmly established that proteins, specifically synthesized in the liver and passed through the kidney into urine, are critical to modulation of urinary signals. The stage
Acknowledgments
Our work is supported by the Biotechnology and Biological Sciences Research Council. We are grateful to Sarah Cheetham, Caroline Payne and Nick Malone for their data on protein:creatinine ratios in inbred and wild mice, to Karen Sanders for providing the data in Fig. 1 and to Duncan Robertson for Fig. 2.
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