BIOCHEMICAL AND PROTEOMIC STUDIES During courtship, a male P. shermani salamander delivers pheromones from a submandibular mental gland to the female’s nares. The pheromones are only delivered after courtship has commenced and their purpose is to increase female receptivity and mating success.  These pheromones do not serve as a sexual attractant.

Biochemical investigations of the glandular extract demonstrated that the pheromone mixture consists of two major families, each of which contains multiple isoforms (Fig. 1).  The 22 kDa family (Plethodontid Receptivity Factor; PRF) is related to the IL-6 family of cytokines, while the 7 kDa family (Plethodontid Modulating Factor; PMF) is related to xenoxins and snake toxins.  

The 22kDa PRF pheromones have been purified, characterized, sequenced, shown to bind to receptors in the vomeronasal organ (VNO) and to increase female receptivity.  A recombinant PRF (rPRF) identical to one of the major natural isoforms has been prepared and shown to possess biological activity similar to the natural isoform.  Recent CD (circular dichroism) experiments of 3 PRF isoforms and rPRF indicate a secondary structure composition that is predominantly alpha-helix with an amount typical of the 4 alpha-helix bundle of IL-6 cytokines.  The 3-dimensional structure of the rPRF will be determined.

The 7 kDa PMF family contains more than 30 very negatively charged isoforms representing the products of multiple genes and allelic variants. The total amounts of various isoforms are consistent from year-to-year, but differences in the relative levels of individual isoforms suggest their expression is differentially regulated at the transcriptional and/or translational levels. 

Recently, we have purified and performed peptide mass fingerprinting of 16 PMF isoforms, of which 8 matched our current database of 32 unique cDNA sequences (Fig. 2). The cDNA library was prepared from mRNA obtained from the mental glands of male P. shermani during their courtship season.  The 7 kDa pheromones have 8 conserved Cys residues (4 disulfide bonds; ~60 residues) with spacing similar to that for xenoxins and snake toxins, but with limited sequence homology.

Conditions for the determination of the disulfide bonding pattern were developed for a single purified isoform which resulted in the average reduction and alkylation of 1, 2 or 3 disulfide bonds. Aliquots of each were then subjected to complete digestion with trypsin, chymotrypsin or Glu-C prior to direct MALDI-TOF analysis. All of the MS data sets are consistent with a disulfide bonding pattern of 1-2, 3-6/7, 4-5, and 6/7-8, with the 3-6/7 bond being more susceptible to reduction and alkylation than the 1-2 bond.  This pattern will dictate a 3-dimensional structure different from that for xenoxins and snake toxins (1-3, 2-4, 5-6, 7-8) and may represent a sub-family for courtship pheromones or a new family of 8 Cys/7 kDa proteins.

MOLECULAR EVOLUTION  Comparative studies of cDNA sequences for PRF and PMF in the genus Plethodon reveal that both proteins have undergone rapid, selection-driven evolution over the last 27 million years.  This continual, rapid evolution is characterized by convergent evolutionary events, gene duplication, production of pseudogenes, and the maintenance of polymorphism (Watts et al. 2004; Palmer et al. 2005, 2006).  We have referred to the process producing this evolutionary pattern in male courtship pheromones as a ‘molecular tango’ and have hypothesized that it is driven by coevolution with receptors in the female VNO (Watts et al. 2004, Palmer et al. 2005).

To test this coevolutionary hypothesis we prepared cDNA from female VNO tissue and used RT-PCR and 5’-RACE PCR to amplify large segments of pheromone receptors.  This strategy revealed 67 distinct V2R isoforms, belonging to at least 6 families, in P. shermani.  Ongoing discovery of new isoforms shows that the V2R family is as diverse in Plethodon as it is in rodents and other vertebrates.  Our preliminary studies in Plethodon have also documented a type of receptor not previously reported from the VNO.  These gp130 and related receptors may mediate PRF reception in the VNO because these receptors are universally utilized by those cytokines most similar to PRF (Watts et al. 2004, Bravo & Heath 2000).  Both gp130 and CNTF-r from Plethodon VNO show unusually high levels of sequence diversity compared to other animals.  This departure from the usual purifying selection mode for gp130 receptors and cytokines is consistent with a coevolutionary process driving diversification of PRF and these gp130 receptors.

We have developed a bacterial expression system for PRF and shown that the recombinant pheromone produced by this system enhances female sexual receptivity (Houck et al. 2006b). Soluble recombinant PRF was expressed in liquid culture with remarkable efficiency (~75mg/L) and was purified using a cleavable metal-affinity tag (6xHis) followed by HPLC. The purified protein is identical to the native isoform, as assayed by mass spectroscopy, circular dichroism measurements of secondary structure, and tests for behavioral effects on female salamanders.  Our ability to produce pure pheromones in quantity sets the stage for the physiological and behavioral bioassays for activity, site-directed mutagenesis studies, and X-ray crystallographic studies of pheromone structure that are included in the present proposal.